pPIC3.5K - Thermo Fisher Scientific

pPIC3.5K/pAO815
Pichia vectors for multicopy
integration and intracellular expression
Catalog no. V173-20, V180-20
Rev. Date: 14 July 2010
Manual part no. 25-0156
MAN0000038
User Manual
ii
Contents
Kit Contents and Storage............................................................................................................................ v
Introduction .............................................................................................................. 1
Product Overview ........................................................................................................................................1
Methods .................................................................................................................... 6
Cloning into pPIC3.5K and pAO815..........................................................................................................6
Analyzing E. coli Transformants...............................................................................................................11
pAO815–In Vitro Multimerization Protocol............................................................................................12
Transformation into Pichia ........................................................................................................................23
pPIC3.5K–In Vivo Screening of Multiple Inserts ....................................................................................27
Appendix ................................................................................................................ 33
Vectors .........................................................................................................................................................33
pPIC3.5K......................................................................................................................................................34
pAO815 ........................................................................................................................................................35
Recipes .........................................................................................................................................................36
Pichia Genomic DNA Isolation .................................................................................................................37
Easy-DNA™ Protocol for Isolation of DNA from Pichia........................................................................39
Determining Copy Number of Multiple Integrants ..............................................................................41
Accessory Products ....................................................................................................................................44
Technical Support.......................................................................................................................................45
Purchaser Notification ...............................................................................................................................46
References....................................................................................................................................................47
iii
iv
Kit Contents and Storage
Shipping and
Storage
pPIC3.5K and pAO815 vectors are shipped on wet ice. Upon receipt, store vectors
at –20°C.
Kit Contents
All vectors are supplied as detailed below. Store the vectors at –20°C.
Catalog no.
Vector
Composition
Amount
V173-20
pPIC3.5K
40 L of 0.5 g/L vector in 10 mM Tris-HCl,
1 mM EDTA, pH 8.0
20 g
V180-20
pAO815
40 L of 0.5 g/L vector in 10 mM Tris-HCl,
1 mM EDTA, pH 8.0
20 g
Intended Use
For research use only. Not intended for any animal or human therapeutic or
diagnostic use.
v
Kit Contents and Storage, Continued
Materials Supplied For the procedures described in this manual, you will need:
by the User
 Manual from the Pichia Expression System
Important
vi

Microbiological equipment

Electrocompetent or chemically competent E. coli (must be recA, endA) for
transformation (page 44). You need 3–4 tubes of competent cells per
experiment. For protocols to prepare competent E. coli and transformation
protocols, see Current Protocols (Ausubel, et al., 1990) or Molecular Biology:
A Laboratory Manual (Sambrook, et al., 1989)

EcoR I, BamH I and Bgl II restriction enzymes and appropriate buffers

Agarose and low-melt agarose

S.N.A.P.™ Gel Purification Kit (see page 44 ) or glass milk

Sterile water

CIP (calf intestinal phosphatase, 1 unit/L)

10X CIP Buffer

Phenol/chloroform

3 M sodium acetate

100% ethanol

80% ethanol

T4 Ligase (2.5 units/L)

10X Ligation Buffer (with ATP)

LB medium

LB-ampicillin plates (50–100 g/mL ampicillin)

16°C, 37°C, and 65°C water baths or temperature blocks

Geneticin® antibiotic (see page 44)

YPD-Geneticin® plates (see Recipes, page 36)

50 mL conical centrifuge tubes

Hemacytometer

30°C and 37°C incubator

Microtiter plates (optional)
Registered Pichia users should already have the Pichia Expression System and
the current manual. Procedures for transformation into E. coli and Pichia,
analysis of recombinants, and expression are described in the Pichia manual.
Introduction
Product Overview
Description of the
System
Multiple copy integration of recombinant genes in Pichia has been demonstrated
to increase expression of the desired gene in some cases (Brierley, et al., 1994;
Clare, et al., 1991a; Cregg, et al., 1993; Romanos, et al., 1991; Scorer, et al., 1993;
Scorer, et al., 1994; Thill, et al., 1990; Vedvick, et al., 1991). The two vectors
included in this kit allow isolation and generation of multicopy inserts by in vivo
or in vitro methods to test whether increasing the copy number of your
recombinant gene will lead to a subsequent increase in protein expression. The in
vivo method utilizes hyper-resistance to Geneticin® (G418 sulfate) to screen for
possible multicopy inserts, while the in vitro method produces tandem inserts of
your gene by ligation.
Frequency of
Multicopy Inserts
Multiple plasmid integration events occur spontaneously in Pichia at a frequency
between 1 and 10% of all His+ transformants. The in vivo method allows you to
screen for the His+ transformants that may have multiple inserts of your gene. The
in vitro method allows you to construct multimers by ligation. When His+
transformants are selected, they have a high probability of containing the
multimers that you constructed in vitro.
Generating
Multicopy Inserts
in vivo
pPIC3.5K contains the bacterial kanamycin gene (kan from Tn903) that confers
resistance to Geneticin® in Pichia. Note that kan does not confer resistance to
kanamycin in Pichia. The level of Geneticin® resistance roughly depends on the
number of kanamycin genes integrated. A single copy of pPIC3.5K integrated
into the Pichia genome confers resistance to Geneticin® to a level of
~0.25 mg/mL. Multiple integrated copies of pPIC3.5K can increase the
Geneticin® resistance level from 0.5 mg/mL (1–2 copies) up to 4 mg/mL (7–12
copies). Because of the genetic linkage between the kanamycin gene and the
"expression cassette" (PAOX1 and your gene of interest), one can infer that
Geneticin® resistant clones contain multiple copies of your gene. Protein
expression may increase because of a gene dosage effect. Thus, the presence of
the kan gene on pPIC3.5K can be used as a tool to detect pPIC3.5K transformants
that harbor multiple copies of your gene. The graphic on the following page
shows multiple insertion and linkage of the kan gene to your expression cassette.
Continued on next page
1
Product Overview, Continued
TT G
ene
of
HIS
4
est
ter
In
5´ PAOX1
Ka
n
3´ AOX1
5´
AOX1 or aox1::ARG4
TT
3´
(
5' PAOX1 Gene of Interest TT
Kan
HIS4
(
3' AOX1
Expression Cassette 1
2nd Insertion Event
5´
AOX1 or aox1::ARG4
TT
3´
(Expression
Cassette 1 (
3' AOX1
(
5' PAOX1 Gene of Interest TT
Kan
HIS4
(
3' AOX1
Expression Cassette 2
3rd Insertion Event, etc.
Screening on
Geneticin®
Direct selection of Geneticin® resistance in yeast does not work well because
newly transformed cells need time to express sufficient amounts of the
resistance factor. Since yeast grow much more slowly than bacteria, significant
numbers of recombinant yeast are killed before they accumulate enough of the
resistance factor to survive direct plating on antibiotic. Do not use Geneticin®
resistance as a selectable marker. The procedure to generate Geneticin®
resistant clones requires an initial selection of His+ transformants followed by a
screen for varying levels of Geneticin® resistance. Resistance to Geneticin®
conferred by the kanamycin gene present on pPIC3.5K is used as a SCREEN, not
as a SELECTION for multicopy integrants.
Continued on next page
2
Product Overview, Continued
The graphic below shows how pAO815 is used to generate multiple expression
cassette copies in a single vector prior to transformation into Pichia. The gene of
interest is inserted into the vector at a unique EcoR I site. The resulting
expression cassette (the PAOX1 plus your gene) is flanked on the upstream side
by a unique Bgl II site and on the downstream site by a unique BamH I site (see
A below).
Generating
Multicopy Inserts
in vitro
The vector containing the gene of interest is digested with Bgl II and BamH I to
excise the expression cassette. The cassette is then reinserted at the BamH I site to
create a tandem repeat of the cassette. The reinsertion process can be repeated to
generate a series of vectors that contain an increasing number of cassettes linked
to a single HIS4 gene (see B below).
Transformation of Pichia with these in vitro-formed multimers increases the
frequency of multicopy expression cassette recombinants. Pichia recombinants
may be custom-designed to contain a defined number of multicopy inserts. For
more information, see page 12.
Bgl II
A.
Vector
EcoR I
5' AOX1 PAOX1
EcoR I BamH I
Gene of Interest
TT
HIS4
1 Expression Cassette
Digestion
with BamH I
Bgl II
B.
Recombinant
Vector
1 Expression
Cassette
Bgl II
Insert
5' AOX1 PAOX1
GATC C
G
HIS4
BamH I
G
GATC T Expression Cassette C CTAG
A
Bgl II
Recombinant
Vector
BamH I
BamH I
G
Expression Cassette C CTAG
BamH I/Bgl II
Gene of Interest
TT
5' AOX1 PAOX1
BamH I
Gene of Interest
TT
HIS4
2 Expression
Cassettes
Continued on next page
3
MEND
ION
AT
RECOM
Product Overview, Continued
We recommend trying both methods to generate or isolate multicopy inserts of
your gene. A summary of the advantages and disadvantages of each method is
presented in the lists below. The "best" method is the one that works for your
protein; unfortunately, there is no way to predict beforehand which method will
work for your protein.
In vivo Method (pPIC3.5K)
Advantages
Disadvantages
•
Easier to initiate experiment because only •
one copy of your gene is cloned into
pPIC3.5K before transforming into Pichia.
Qualitative screen – Geneticin® resistance
may not necessarily correlate with the
number of copies of your gene.
•
Identifies the 1–10% of spontaneous His+
transformants that have multiple inserts.
•
Screening His+ transformants may involve
more work because you need thousands of
His+ transformants to generate enough
Geneticin® resistant colonies to test.
•
Average size of vector is similar to other
Pichia expression vectors.
•
The number of multiple inserts is unknown
(although this can be determined through
Southern or dot blot analysis).
•
Multiple inserts are located at a single
locus.
•
Screening on Geneticin® is sensitive to the
density of the cells and may result in the
isolation of false positives.
In vitro Method (pAO815)
Advantages
Disadvantages
•
Quantitative – construction of a defined
number of multimers.
•
More work up front to clone defined
number of multimers.
•
Most of the His+ transformants will
contain the proper, defined number of
inserts.
•
Size of the vector may become quite large
depending on the size of your gene and the
number of copies you create.
•
Isolation of recombinants with multiple
inserts is easier because most of the His+
transformants contain multiple copies of
your gene.
•
Rearrangements in E. coli may occur.
•
In vitro construction allows step-wise
analysis of copy number effects on
protein expression.
•
Multiple inserts are located at a single
locus.
•
No need for a second drug resistance
marker in the vector.
Continued on next page
4
Product Overview, Continued
pPIC3.5K
pPIC3.5K is a plasmid designed to allow you to identify in vivo multiple
integrations of your gene in the Pichia genome.

9004 bp vector

Five unique restriction sites in the multiple cloning site: BamH I, SnaB I,
EcoR I, Avr II, Not I

Intracellular expression of your gene

Requires an initiating ATG codon in a Kozak consensus sequence for proper
translation initiation of your gene (Cavener and Stuart, 1991; Kozak, 1987;
Kozak, 1990)

HIS4 selection in Pichia

For insertion at AOX1 in GS115 or KM71, linearize with Sac I (generates His+
Mut+ in GS115 and His+ MutS in KM71)

For insertion at HIS4, linearize with Sal I (generates His+ Mut+ in GS115 and
His+ MutS in KM71)

For a gene replacement at AOX1 in GS115, linearize with Bgl II (generates
His+ MutS)
See page 24 for alternate restriction sites if your insert DNA has a Bgl II, Sac I, or
Sal I site.
pAO815
pAO815 is a plasmid designed for in vitro generation of multimers of your gene
for integration into the Pichia genome.

7709 bp vector

One unique restriction site: EcoR I

Intracellular expression of your gene

Requires an initiating ATG codon in a Kozak consensus sequence for proper
translation initiation of your gene (Cavener and Stuart, 1991; Kozak, 1987;
Kozak, 1990)

HIS4 selection in Pichia

For insertion at HIS4, linearize with Sal I or Stu I (generates His+ Mut+ in
GS115 and His+ MutS in KM71)

For a gene replacement at AOX1 in GS115, linearize with Bgl II (generates
His+ MutS)
See page 24 for alternate restriction sites if your insert DNA has a Bgl II, Stu I, or
Sal I site.
5
Methods
Cloning into pPIC3.5K and pAO815
MEND
For help with DNA ligations, E. coli transformations, restriction enzyme analysis,
purification of single-stranded DNA, DNA sequencing, and DNA biochemistry,
refer to Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989) or Current
Protocols in Molecular Biology (Ausubel et al., 1994) (See References, page 47).

We recommend that you ligate your insert into both pPIC3.5K and pAO815
so that you can try both methods to isolate multiple integrants. Below are
some guidelines to consider when developing a cloning strategy for these
vectors. The multiple cloning sites for each vector are presented on the
pages 9–10 for your convenience.

We recommend that you transform the two supercoiled Pichia expression
vectors into E. coli, so that you have a permanent stock and a way to make
more plasmid. Transform competent E. coli with 1–2 L of the supplied
vector stock solution and select on LB with 50–100 g/mL ampicillin (LBAmp).
ION
AT
RECOM
General Molecular
Biology
Techniques
General
Considerations
The following general considerations are applicable to both vectors.

The codon usage in Pichia is believed to be the same as Saccharomyces
cerevisiae.

Many Saccharomyces genes have proven to be cross-functional in Pichia.

Maintain plasmid constructions in a recA, endA mutant E. coli strain such as
TOP10. Electrocompetent TOP10 cells are available from Invitrogen (see
page 44).

The native 5´ end of the AOX1 mRNA is noted in each multiple cloning site.
This is needed to calculate the size of the expressed mRNA of the gene of
interest if you need to analyze mRNA for any reason.

Translation termination is determined by either stop codons in the gene of
interest or in the 3´ AOX1 sequence. The stop codons in the 3´ AOX1
sequence are noted in each figure on pages 9–10.

The premature termination of transcripts because of "AT rich regions" has
been observed in Pichia and other eukaryotic systems (Henikoff and Cohen,
1984; Irniger, et al., 1991; Scorer, et al., 1993; Zaret and Sherman, 1984). If you
have problems expressing your gene, check for premature termination and
AT rich regions. It may be necessary to change the sequence to express your
gene (Scorer, et al., 1993).
Continued on next page
6
Cloning into pPIC3.5K and pAO815, Continued
General Cloning
Strategies
Strategies generally fall into three different categories:
1.
Ligation of a compatible restriction fragment:
a.
Forced (directional) insertion involving the use of two different sites in
the multiple cloning site (pPIC3.5K).
b. Ligation of the fragment with the same restriction end on both ends into
a single, compatible site (e.g. EcoR I cloning in pAO815). Note that you
will need to dephosphorylate pAO815 to ligate into the EcoR I site.
Important
2.
PCR amplification of the fragment containing the gene of interest in such a
way that compatible restriction ends are generated for ligation into the
appropriate vector.
3.
Direct cloning of an amplified fragment containing the gene of interest via
the TA Cloning® Kit (see page 44) followed by subcloning of a compatible
fragment into the appropriate Pichia expression vector.
If your insert has an EcoR I site and you are trying to clone into the EcoR I site of
pAO815, we recommend the following:
1.
An enzyme like Bsa I has the following restriction recognition site:
5´-GGTCTCNˇ
3´-CCAGAGNNNNNˆ
2.
An EcoR I site may be engineered into the recognition site for Bsa I.
5´-GGTCTCGˇAATTC.....
3´-CCAGAGCTTAAˆG.....
3.
This sequence may be added to your DNA fragment by integrating it into
your PCR primer or created in vitro as an adaptor to another restriction site.
4.
Digest your PCR or adapted ligation product with Bsa I. This will generate
EcoR I overhangs on both ends of your fragment without digesting with
EcoR I.
5´- AATTC......
3´-G......
5.
Ligate into dephosphorylated pAO815. Other enzymes that may be used are
BsmA I or BsmB I.
Continued on next page
7
Cloning into pPIC3.5K and pAO815, Continued
Cloning
Procedures
Refer to (Ausubel, et al., 1990), pages 3.16.1–3.17.3. or (Sambrook, et al., 1989),
pages 5.10–5.13. for help with cloning.
Bacterial
Transformation
Once you have decided on a cloning strategy, you can use electrocompetent or
chemically competent E. coli for transformation.
Continued on next page
8
Cloning into pPIC3.5K and pAO815, Continued
PAOX1 and Multiple
Cloning Site of
pPIC3.5K
The sequence below shows the details of the multiple cloning site and
surrounding sequences.
AOX1 mRNA 5´ end (824)
5´ AOX 1 Primer Site (855-875)
TTATCATCAT TATTAGCTTA CTTTCATAAT TGCGACTGGT TCCAATTGAC
AAGCTTTTGA TTTTAACGAC TTTTAACGAC AACTTGAGAA GATCAAAAAA
BamH I
SnaB I
EcoR I
Avr II
Not I
CAACTAATTA TTCGAAGGAT CCTACGTAGA ATTCCCTAGG GCGGCCGCGA
ATTAATTCGC CTTAGACATG ACTGTTCCTC AGTTCAAGTT GGGCACTTAC
3´ AOX 1 Primer Site (1055-1075)
GAGAAGACCG GTCTTGCTAG ATTCTAATCA AGAGGATGTC AGAATGCCAT
TTGCCTGAGA GATGCAGGCT TCATTTTTGA TACTTTTTTA TTTGTAACCT
AOX1 mRNA 3´ end (1146)
ATATAGTATA GGATTTTTTT TGTCATTTTG TTTCTTC
Special
Considerations

For pPIC3.5K, the fragment containing the gene of interest should have a
Kozak consensus sequence for proper translation initiation, although this
requirement is not as stringent in yeast. For example, ACC ATG G is a
Kozak consensus sequence, where the ATG corresponds to the initiating
ATG for your gene of interest (Cavener and Stuart, 1991; Kozak, 1987; and
Kozak, 1990). Note: There is an ATG upstream of the SnaB I site.

Be sure to analyze the 5´ untranslated region of the mRNA for secondary
structure formation. Secondary structure in the mRNA may have a negative
effect on expression of the recombinant protein.

If you are digesting with BamH I and SnaB I or SnaB I and EcoR I, digest with
SnaB I first. If you digest with BamH I or EcoR I first, the SnaB I site will be
too close to the end of the DNA and will not be digested properly.
Continued on next page
9
Cloning into pPIC3.5K and pAO815, Continued
PAOX1 and Multiple
Cloning Site of
pAO815
The sequence below shows the details of the multiple cloning site and
surrounding sequences.
AOX1 mRNA 5´ end (824)
5´ AOX 1 primer site (855-875)
TTATCATCAT TATTAGCTTA CTTTCATAAT TGCGACTGGT TCCAATTGAC
AAGCTTTTGA TTTTAACGAC TTTTAACGAC AACTTGAGAA GATCAAAAAA
EcoR I
CAACTAATTA TTCGAAACGA GGAATTCGCC TTAGACATGA CTGTTCCTCA
GTTCAAGTTG GGCACTTACG AGAAGACCGG TCTTGCTAGA TTCTAATCAA
3´ AOX 1 primer site (1024-1044)
GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT CATTTTTGAT
AOX1 mRNA 3´ end (1115)
ACTTTTTTAT TTGTAACCTA TATAGTATAG GATTTTTTTT GTCATTTTGT
Special
Considerations
Transformation
10

For in vitro multimerization, you need to analyze your insert for BamH I and
Bgl II restriction sites. If your insert has a BamH I or Bgl II site, we
recommend that you use the in vivo method (pPIC3.5K) to isolate multiple
inserts of your gene.

For pAO815, the fragment containing the gene of interest should have a
Kozak consensus sequence for proper translation initiation, although this
requirement is not as stringent in yeast. For example, ACC ATG G is a
Kozak consensus sequence, where the ATG corresponds to the initiating
ATG for your gene of interest (Cavener and Stuart, 1991; Kozak, 1987; and
Kozak, 1990).

Be sure to analyze the 5´ untranslated region of the mRNA for secondary
structure formation. Secondary structure in the mRNA has a negative effect
on expression of the recombinant protein.
At this point you should have ligation reactions that you will transform by
chemical means or electroporation into competent E. coli cells such as TOP10
(see page 44 for ordering) using your method of choice.
Analyzing E. coli Transformants
Analyzing
Transformants
Sequencing
Recombinant
Clones
1.
After transformation, plate 10 L and 100 L of the transformation mix onto
LB plates with 50–100 g/mL ampicillin and select ampicillin resistant
colonies.
2.
Pick 10 ampicillin resistant transformants and inoculate into 2 mL LB
medium with 50–100 g/mL ampicillin. Grow overnight at 37°C with
shaking.
3.
Isolate plasmid DNA by miniprep for restriction analysis and sequencing
(see below). To sequence the Pichia expression vectors (pPIC3.5K and
pAO815 with only one insert), use the 5´ AOX1 and the 3´ AOX1
sequencing primers.
4.
Make a glycerol stock of your desired clone by combining 0.85 mL of a
overnight bacterial culture with 0.15 mL of sterile glycerol. Mix by vortexing
and transfer to a labeled storage tube. Freeze the tube in liquid nitrogen or a
dry ice/ethanol bath and store at –80°C.
We strongly recommend that you sequence your construct in pPIC3.5K and
pAO815 (with only one insert) before proceeding further to confirm that the
ATG is in the proper context for eukaryotic translation initiation. Use the 5´ and
3´ AOX1 sequencing primers to sequence your constructs.
Sequencing primers are available from Invitrogen (see page 44).
After Sequencing
Once you have cloned and sequenced your insert, proceed as directed below:

If you cloned your insert into pAO815, proceed to In Vitro Multimerization,
next page.

If you cloned your insert into pPIC3.5K, generate enough plasmid DNA to
transform Pichia (5–10 g of each plasmid per each transformation). Proceed
to Transformation into Pichia, page 23.
11
pAO815–In Vitro Multimerization Protocol
Introduction
At this point you should have your gene cloned into the EcoR I site of pAO815
(recombinant pAO815). You will use this vector for two purposes. First you will
use it to generate a Bgl II-BamH I expression cassette consisting of the AOX1
promoter and your gene. Second, you will linearize the vector using BamH I to
allow cloning of multiple copies of the Bgl II-BamH I expression cassette. Note
that the linearized vector already contains one copy of your expression cassette.
To generate multiple copies of your expression cassette:
Step
Description
1
Treat your Bgl II-BamH I expression cassette with ligase in vitro. Note
that Bgl II and BamH I share 4 bases in common between their
recognition sites.
2
Generate head-to-tail, head-to-head, and tail-to-tail multimers
(Head-to-tail ligation, which is the correct orientation for expression,
will destroy both the BamH I and Bgl II sites).
3
Treat the ligation mix with BamH I and Bgl II to eliminate head-tohead and tail-to-tail multimers.
4
Ligate into BamH I-linearized recombinant pAO815.
5
Transform into E. coli and analyze recombinant plasmids for copy
number by digesting with Bgl II and BamH I.
Continued on next page
12
pAO815–In Vitro Multimerization Protocol, Continued
The figure below and on the following page outlines the multimerization
process.
EcoR I
EcoR I
o f I n t er
est
Digest with
BamH I
4
Amp
i ci l
Recombinant
pAO815
BamH I
l in
HIS
Bgl II
G ene
X
O
TT
5´
A
Flow Chart of
Multimerization
Process
3´ AOX1
Bgl II
Digest with
BamH I/Bgl II
Bgl II
PO4
BamH I
Gene of Interest
5' AOX1 PAOX1
TT
PO4
Expression Cassette
Treat w/
Ligase
Bgl II
BamH I/Bgl II*
Expression Cassette
PO4
BamH I
Expression Cassette
PO4
head-to-tail
BamH I
Bgl II
Bgl II
Expression Cassette
PO4
Expression Cassette
PO4
tail-to-tail
BamH I
PO4
Bgl II
BamH I
Expression Cassette
PO4
Expression Cassette
head-to-head
Digest w/
BamH I
and Bgl II
Bgl II
PO4
BamH I/Bgl II*
Expression Cassette
Bgl II
BamH I
BamH I
PO4
BamH I
Expression Cassette
PO4
PO4
Bgl II
Expression Cassette
Bgl II
Expression Cassette
BamH I
Expression Cassette
Bgl II
PO4
BamH I
Expression Cassette
PO4
* Site is not cleavable by BamH I or Bgl II
Continued on next page
13
pAO815–In Vitro Multimerization Protocol, Continued
Flow Chart of Multimerization Process, Continued
BamH I
PO4
Bgl II
HIS4
3´ AOX1
Amp
BamH I
5´ AOX1 PAOX1
Gene of Interest
TT
PO4
Dephosphorylate
the Vector
BamH I
Bgl II
HIS4
3´ AOX1
Amp
5´ AOX1 PAOX1
BamH I
Gene of Interest
TT
BamH I/Bgl II
of
X1 Gene Interest
5´AO
Recombinant
pAO815
BamH I
c
pi
Am
IS
4
5´AOX1 G
ene
of
Bgl II
5´A
OX
1
TT
Mix Vector
and Inserts
Ligate
BamH I/Bgl II
TT
t
teres
of In
ne
Ge
Inte
res
t
TT
i ll
in
H
Sal I
Stu I
3 ´ AO X 1
Bgl II
Continued on next page
14
pAO815–In Vitro Multimerization Protocol, Continued
Alternative
Procedure
You may wish to build each desired multimer in increments by ligating each
additional expression cassette one (or two) at a time into pAO815. For example:
Step
Materials Needed
Description
1
Digest pAO815 with one copy of your gene using BamH I.
2
Ligate a single copy of the Bgl II-BamH I expression cassette into the
vector.
3
Transform E. coli and analyze the transformants for the vector with
2 copies of your insert.
4
Isolate and digest this vector (with 2 copies of your gene) with
BamH I and Bgl II to isolate a cassette with 2 copies of your gene
(optional).
5
Digest the vector with 2 copies of your gene with BamH I and ligate
1 or 2 copies (see Step 4) of the expression cassette into the vector.
6
Transform E. coli and analyze the transformants for the vector with
3 or 4 copies of your insert.
7
Repeat until the desired multimer is reached.

Electrocompetent or chemically competent E. coli (must be recA, endA) for
transformation. You will need 3–4 tubes of competent cells per experiment.
See page 44 for ordering information.

EcoR I, BamH I and Bgl II restriction enzymes and appropriate buffers

Low-melt agarose

S.N.A.P.™ Gel Purification Kit (see page 44) or glass milk

Sterile water

CIP (calf intestinal alkaline phosphatase, 1 unit/L)

10X CIP Buffer

Phenol/chloroform

3 M sodium acetate

100% ethanol

80% ethanol

T4 Ligase (2.5 units/L)

10X Ligation Buffer (with ATP)

LB-Amp plates (50–100 g/mL ampicillin)

16°C, 37°C, and 65°C water baths or temperature blocks
Continued on next page
15
pAO815–In Vitro Multimerization Protocol, Continued
Controls
To evaluate your transformants and expression data later on, we recommend
transforming Pichia with pAO815 (the parent vector) and pAO815 containing
one copy of your expression gene. This will allow you to compare expression
levels to see if multiple copies significantly increase the amount of protein
produced. Also, if you elect to determine how many copies of your gene are in a
recombinant by dot or Southern blot, the strain with the parent vector will
control for background hybridization and the strain with the single copy gene
will provide a signal to normalize your data.
Digesting
Recombinant
pAO815
Set up two separate digestions of recombinant pAO815 containing one copy of
your gene:
Producing
Expression
Cassettes for
Multimerization
1.
Double digest 1–2 g recombinant pAO815 with 10 units each of Bgl II and
BamH I. Use a 20 L reaction volume and digest for 1–2 hours at 37°C to
release your expression cassette. Proceed to Producing Expression Cassettes
for Multimerization, Step 1.
2.
Digest 2 g recombinant pAO815 with 10 units of BamH I only. Use a 20 L
reaction volume and digest for 1–2 hours at 37°C to linearize recombinant
pAO815. Proceed to Dephosphorylating the Vector, Step 1.
The S.N.A.P.™ Gel Purification Kit available from Invitrogen (see page 44 for
ordering) allows you to rapidly purify DNA fragments from regular agarose
gels. Alternatively, you may use glass milk. To use the S.N.A.P.™ Gel
Purification Kit, follow the steps below:
1.
Electrophorese your digest from Step 1, above, on a 1 to 5% regular TAE
agarose gel. Note: Do not use TBE to prepare agarose gels. Borate interferes
with the sodium iodide step, below.
2.
Cut out the gel slice containing the PCR product and melt it at 65°C in
2 volumes of the 6 M sodium iodide solution.
3.
Add 1.5 volumes Binding Buffer.
4.
Load solution (no more than 1 mL at a time) from Step 3 onto a S.N.A.P.™
column. Centrifuge 1 minute at 3,000 × g in a microcentrifuge and discard
the supernatant.
5.
If you have solution remaining from Step 3, repeat Step 4.
6.
Add 900 L of the Final Wash Buffer.
7.
Centrifuge 1 minute at full speed in a microcentrifuge and discard the flowthrough.
8.
Repeat Step 7.
9.
Elute the purified DNA in 15 L of sterile water. Store on ice if proceeding
immediately to Ligating the Expression Cassette, page 18. Store at –20ºC for
long-term storage.
Continued on next page
16
pAO815–In Vitro Multimerization Protocol, Continued
Dephosphorylating Dephosphorylation is necessary to prevent self-ligation of the vector.
the Vector
1. Take your digest from Digesting Recombinant pAO815, Step 2 and phenol
extract, then ethanol precipitate the DNA. Resuspend in 17 L of sterile
water.
2.
Set up the dephosphorylation reaction in a microcentrifuge tube as follows:
BamH I digested recombinant pAO815
(page 16, Step 2)
17 L
10X CIP Buffer
2 L
CIP (1 Unit/L)
1 L
Total Volume
20 L
3.
Incubate at 37°C for 15 minutes.
4.
Add 30 L sterile water to the reaction to make a final volume of 50 L.
5.
Add 50 L phenol/chloroform and extract your DNA solution. Transfer the
aqueous solution to a new tube.
6.
Precipitate the DNA by adding 5 L 3 M sodium acetate and 110 L 100%
ethanol. Incubate on ice for 30 minutes.
7.
Centrifuge at maximum speed in a microcentrifuge for 10 minutes at 4°C.
Carefully decant the supernatant.
8.
Wash the nucleic acid pellet with 80% ethanol, centrifuge 2 minutes, and
remove the ethanol.
9.
Centrifuge again for 1 minute, remove residual ethanol, and air dry the
pellet.
10. Resuspend the pellet in 8 L sterile water. Save on ice if you plan to ligate
your insert immediately (see Ligating and Digesting the Expression
Cassette, next page) or store at –20°C.
Continued on next page
17
pAO815–In Vitro Multimerization Protocol, Continued
Ligating and
Digesting the
Expression
Cassette
Ligation of the expression cassette will generate head-to-tail, head-to-head, and
tail-to-tail multimers. Creation of head-to-tail multimers will be in the correct
orientation for expression and will destroy both the BamH I and Bgl II sites
between the expression cassettes. Digestion of the multimers with BamH I and
Bgl II will eliminate those multimers with tail-to-tail and head-to-head
orientation. After digestion with these two restriction enzymes, you will have a
mixture of multimers containing 1, 2, 3, etc. copies of your gene that can be
ligated into BamH I-linearized, recombinant pAO815.
1.
Set up a 20 L ligation reactions as follows:
Bgl II-BamH I digested expression cassette
15 L
Sterile water
2 L
10X Ligation Buffer (with ATP)
2 L
T4 DNA Ligase (2.5 units/L)
1 L
Total Volume
20 L
2.
Incubate at 16°C for 2.5 hours.
3.
Heat inactivate the ligase by incubating at 65°C for 20 minutes.
4.
Add the following reagents for restriction enzyme digestion (cut-back). Note
that BamH I and Bgl II may be used with the same restriction buffer:
Sterile water
10X restriction enzyme buffer
23 L
5 L
Bgl II (10 units/mL)
1 L
BamH I (10 units/mL)
1 L
Total Volume
30 L
5.
Incubate the reaction at 37°C for 2 hours.
6.
Add 50 L phenol/chloroform and extract the restriction enzyme digestion
to remove the enzymes. Transfer the aqueous solution to a new
microcentrifuge tube.
7.
To ethanol precipitate the DNA, add 5 L 3 M sodium acetate and 110 L
100% ethanol.
8.
Centrifuge at maximum speed in a microcentrifuge for 10 minutes at 4°C.
Carefully decant the supernatant.
9.
Wash the nucleic acid pellet with 80% ethanol, centrifuge 2 minutes, and
remove the ethanol. Centrifuge again for 1 minute, remove residual ethanol,
and air dry the pellet.
10. Resuspend pellet in 4 L sterile water. Save on ice if you plan to ligate your
insert immediately or you can store at –20°C. Proceed to Ligating
Multimers into Linearized Vector, next page.
Continued on next page
18
pAO815–In Vitro Multimerization Protocol, Continued
You may wish to combine the ligation reaction with the restriction enzyme
digestion. T4 ligase will retain most of its activity in all of the four New England
BioLabs buffers. Remember to add 1 mM ATP to the reaction to ensure ligase
activity.
Ligating Multimers You are now ready to ligate the mixture of multimers generated in Step 10,
above, into dephosphorylated, linearized vector.
into Linearized
Vector
1. Set up the following ligation reactions:
Dephosphorylated vector (page 17, Step 10)
4 L
Expression cassette multimers (Step 10, above)
4 L
10X Ligation Buffer
1 L
T4 DNA Ligase (2.5 units/L)
1 L
Total volume
10 L
For the vector only control:
Dephosphorylated vector
4 L
Sterile water
4 L
10X Ligation Buffer
1 L
T4 DNA Ligase (2.5 units/L)
1 L
Total volume
Transforming
E. coli
10 L
2.
Incubate overnight at 16°C.
3.
You may store the ligation reactions at –20°C until ready to use, or
transform 1–10 L of each ligation mix into competent E. coli. Note that the
amount of the ligation mixture you transform depends on whether you use
electrocompetent or chemically competent cells. You may have to decrease
the amount you to transform into electrocompetent cells to prevent arcing.
Remember to include the "vector only" and "cells only" controls to evaluate your
experiment. The "vector only" will indicate whether your vector was
dephosphorylated. Since the CIP reaction is not 100% and because you often get
degradation of the ends, there might be a few colonies on this plate. The "cells
only" plate should have no colonies at all.
1.
Transform competent E. coli by your method of choice.
2.
After adding medium to the transformed cells and allowing them to recover,
plate 10 L and 100 L of each transformation mix onto LB plates with 50–
100 g/mL ampicillin. Save the remainder of your transformation mix at
4°C.
3.
Incubate overnight at 37°C. If you do not get transformants or very few
transformants, plate out the remainder of the transformation mix onto LBampicillin plates.
Continued on next page
19
pAO815–In Vitro Multimerization Protocol, Continued
Analyzing
Transformants
1.
Pick 20 transformants and inoculate 2 mL LB containing 50–100 g/mL
ampicillin. Grow overnight at 37°C.
2.
Isolate plasmid DNA and digest with Bgl II and BamH I to release any
multimers from pAO815.
(Be sure to include Bgl II-BamH I digested pAO815 as a control. It is possible
to get vector rearrangements and deletions with large vectors in E. coli.
Including Bgl II-BamH I digested pAO815 will allow you to detect these
rearrangements-deletions in the vector backbone.)
3.
Analyze your digests on a 1% agarose gel. You should see bands
corresponding to 1 copy, 2 copies, 3 copies, etc. of your expression cassette
along with the vector backbone.
(The number of copies you obtain may depend on how well a large vector is
tolerated by the host strain.)
4.
Once you have identified plasmids with multiple copies of your expression
cassette, be sure to purify by streaking for single colonies and confirming
your construct.
5.
Prepare frozen glycerol stocks of E. coli containing each of your multimeric
constructs.
6.
Prepare 5–10 g of each plasmid for transformation into Pichia. Proceed to
Transformation into Pichia, page 23.
Continued on next page
20
pAO815–In Vitro Multimerization Protocol, Continued
Troubleshooting
The table below will help you optimize formation and isolation of multimers in
Pichia.
Problem
No multimers or low
number of multimers in
your vector after
transformation into E. coli
Possible Reason
CIP defective
Solution
Use fresh CIP.
Add more CIP. Add 1 unit of CIP and
incubate 15 more minutes at 37°C.
This is somewhat risky as CIP can
degrade the ends of your DNA.
Not enough insert DNA to
ligate
Digest more pAO815 containing
1 copy of your expression cassette.
Construct is unstable in
E. coli
Use the in vivo method to isolate
multimers (see page 27).
Multimers are too long to
ligate efficiently
Try ligating each expression cassette
separately.
Recombinant vector
Construct is unstable in
rearranges and deletions are E. coli
detected
Use the in vivo method to isolate
multimers (see page 27).
Pichia His+ transformants do Vector was linearized with
the wrong enzyme.
not have multimers
Restriction enzymes in the
5´ AOX1 region are
duplicated when
multimers are created.
Linearize your construct with Sal I or
Stu I to insert the construct into his4.
Analyze your construct for other
unique restriction sites in the vector
backbone that are near the 5´ AOX1
region or the 3´ AOX1 region. These
sites will preserve your multimers
and allow recombination with AOX1.
Continued on next page
21
pAO815–In Vitro Multimerization Protocol, Continued
For More
Information
There are a number references in the literature you can consult to optimize
synthesis of in vitro multimers. A partial list is provided below:
Cohen, B. and Carmichael, G. G. (1986) A Method for Constructing Multiple
Tandem Repeats of Specific DNA Fragments. DNA 5: 339-343.
Eisenberg, S., Francesconi, S. C., Civalier, C. and Walker, S. S. (1990) Purification
of DNA-Binding Proteins by Site-specific DNA Affinity Chromatography.
Methods Enzymol. 182: 521-529.
Graham, G. J. and Maio, J. J. (1992) A Rapid and Reliable Method to Create
Tandem Arrays of Short DNA Sequences. BioTechniques 13: 780-789.
Rudert, W. A. and Trucco, M. (1990) DNA Polymers of Protein Binding
Sequences Generated by Polymerase Chain Reaction. Nucleic Acids Res. 18:
6460.
Simpson, R. T., Thoma, F. and Brubaker, J. M. (1985) Chromatin Reconstituted
from Tandemly-repeated Cloned DNA Fragments and Core Histones: A
Model System for the Study of Higher-order Structure. Cell 42: 799-808.
Takeshita, S., Tezuka, K.- i., Takahashi, M., Honkawa, H., Matsuo, A., Matsuishi,
T. and Hashimoto-Gotoh, T. (1988) Tandem Gene Amplification in vitro for
Rapid and Efficient Expression in Animal Cells. Gene 71: 9-18.
Taylor, W. H. and Hagerman, P. J. (1987) A General Method for Cloning DNA
Fragments in Multiple Copies. Gene 53: 139-144.
22
Transformation into Pichia
Introduction
At this point you should have your gene cloned as multimers in pAO815 and
singly in pPIC3.5K. You should also have about 5–10 g of each construct for
each transformation into Pichia. For methods to transform Pichia and select His+
transformants, refer to the Pichia Expression System manual. To linearize your
construct prior to transformation into Pichia, see below.
Linearizing
Plasmid DNA
To linearize your construct in pPIC3.5K or pAO815, read the following:
1.
2.
If you cloned your insert into pPIC3.5K, you will need to linearize your
insert prior to transformation using:

Bgl II for replacement at AOX1 (GS115)

Sac I for insertion at AOX1 (GS115 or KM71)

Sal I for insertion at HIS4 (GS115 or KM71)
If you cloned your insert into pAO815, you will need to linearize your insert
prior to transformation using:

Bgl II for replacement at AOX1 (GS115)

Sal I or Stu I for insertion at HIS4 (GS115 or KM71)
Note that multiple Sac I sites are formed if there are 2 or more multimers in
pAO815.
MEND
ION
AT
RECOM
If your insert contains any of these restriction sites, see the table on the next page
for alternate sites.
We recommend that you linearize your vector in such a manner to generate both
Mut+ and MutS recombinants. It is possible that one phenotype will express your
multicopy integrant better than the other.
If you want only Mut+ recombinants:

Linearize pPIC3.5K with Sac I or Sal I for insertion at AOX1 or his4,
respectively, and transform GS115.

Linearize pAO815 with Sal I or Stu I for insertion at his4 and transform
GS115.
If you wish to have only MutS recombinants:

Use strain KM71 which is already MutS and linearize for insertion at AOX1
or his4.

Linearize pPIC3.5K with Bgl II for gene replacement at AOX1 and transform
GS115.

Linearize pAO815 with Bgl II for gene replacement at AOX1 and transform
GS115.
Continued on next page
23
Transformation into Pichia, Continued
Alternate
Restriction Sites
The table below describes alternate restriction sites for linearizing your construct
before transformation into Pichia.
pPIC3.5K. Note that an additional Stu I site was added with the inclusion of the
kan gene, so that the Stu I site in HIS4 is no longer unique.
5´ AOX1
3´ AOX1
Vector backbone
HIS4 gene
Sac I
209
--
--
--
Pme I
414
--
--
--
Bpu 1102 I
589
--
--
--
Xcm I
699
--
--
--
Aat II*
(8843)
--
--
--
Restriction
Enzyme
Tth III I*
--
(6775)
--
--
Bgl
II†
2
6616
--
--
Dra
I†
414
6454
6596, 7787, 7806
--
Sal I
--
--
--
2919
BspE I
--
--
--
3580
pAO815. Note that if more than one expression cassette is created in pAO815,
most of the unique sites in the 5´ AOX1 region are now duplicated and no longer
unique.
Restriction
Enzyme
5´ AOX1
3´ AOX1
Vector backbone
HIS4 gene
(7535)
--
--
--
Tth III I*
--
(5467)
--
--
Bgl II†
2
5307
--
--
Sal I
--
--
--
2863
Stu I
--
--
--
2948
BspE I
--
--
--
3580
Aat II*
*Restriction sites are outside the AOX1 sequences in the vector backbone, but
they are close enough for efficient recombination to occur.
†Restriction sites are used to generate gene replacements at AOX1 in GS115
only.
Continued on next page
24
Transformation into Pichia, Continued
Controls
We recommend that you include the following controls when transforming
Pichia.

The parent vector linearized in the same manner as your construct. This is
used as a control to confirm integration via PCR (see the Pichia Expression
Manual for a protocol) and as control for background for the expression
analysis and the quantitative dot blots or Southern analysis.

pAO815 or pPIC3.5K containing one copy of your expression cassette. Be
sure to linearize pAO815 in the same manner as your multimer. Most of the
His+ transformants created by transforming with recombinant pPIC3.5K will
only have one copy. Make sure that the transformant you pick is only
resistant to 0.25 mg/mL Geneticin®. The single copy controls created using
pPIC3.5K and pAO815 should have the same Mut phenotype as the putative
multimeric recombinants you are testing. These recombinants will be used
as a control to compare expression levels with multiple copies of your
expression cassette and as a single copy control for quantitative dot blot or
Southern analysis. This is a very important control as increasing the copy
number of the desired gene does not always lead to increased expression of
recombinant protein.
Transforming
Pichia
Refer to the Pichia Expression Manual for procedures to prepare Pichia for
transformation, transformation procedures, and selection of His+ recombinants.
Invitrogen also offers the EasyComp™ Kit for preparation and transformation of
competent Pichia cells (see page 44).
Analysis of His+
Transformants
Once you have generated His+ transformants using recombinant pPIC3.5K,
proceed to In Vivo Selection of Multiple Inserts, page 27.
For His+ transformants generated using recombinant pAO815, you will need to
analyze recombinants for the presence of your insert. Refer to PCR Analysis of
Pichia Integrants in the Pichia Expression System manual. Analyze for the
presence of your insert by PCR (see the Pichia Expression System manual for a
protocol). Note: The size of the PCR product for pAO815 is 189 bp.
Important
PCR will only indicate if your gene is present but will not indicate how many
copies of your gene are integrated or at which locus it is integrated. PCR can
reasonably be done on 12–20 transformants. Remember to include vector only and
original construct controls to analyze your PCR experiment.
Since there is no guarantee that multiple copies will actually increase the amount
of protein expressed, most people elect to proceed directly to expression to see if
any of these colonies overexpress their protein. Be sure to include a single copy
insert as a control. Test all your multimeric His+ transformants for their Mut
phenotype so that you induce expression properly. Refer to the Pichia Expression
System manual for methods to express your protein.
Continued on next page
25
Transformation into Pichia, Continued
26
MEND
ION
AT
RECOM
Determining Copy
Number
If you find that your His+ recombinants significantly overexpress your protein,
you may wish to quantify the copy number of your gene. Copy number may be
analyzed by Southern or quantitative dot (slot) blots (see page 41). It is very
important to include genomic DNA isolated from the host strain, Pichia
recombinants transformed with the parent vector, and Pichia recombinants
transformed with pPIC3.5K or pAO815 containing a single copy of your gene as
controls to evaluate your experiment.
Be sure to purify your clones by streaking for single colonies and making frozen,
glycerol stocks of all your Geneticin® resistant colonies. Always initiate
expression studies from frozen stocks, not old plates.
pPIC3.5K–In Vivo Screening of Multiple Inserts
Introduction
You will need as many His+ transformants as you can conveniently generate.
Note that statistically 1–10% of the His+ transformants will have more than one
insert. This means that if the frequency of multicopy inserts is 1%, you will have
to screen 1000 His+ transformants to get 10 Geneticin® resistant colonies to test.
This may require 1–5 plates containing His+ transformants. It is not unusual to
screen thousands of colonies. Once you have Geneticin® resistant colonies, you
can then characterize them for their Mut phenotype.
Methods to Screen There are two methods used to screen His+ transformants for Geneticin®
resistance:
for Geneticin®
Resistant
 Method 1 is technically easier and screens a greater number of clones, but is
Transformants
less reliable. After initial selection of His+ transformants, they are pooled
and plated on YPD-Geneticin® plates containing increasing concentrations of
Geneticin®. Method 1 is applicable to spheroplast or electroporation
transformation methods.

Important
Before Starting
Method 2 is technically more difficult and screens fewer numbers of clones,
but it is more reliable. It involves growing clones in microtiter plates until all
clones are at the same density. The cultures are then spotted on the YPDGeneticin® plates and scored for Geneticin® resistance.
There is a tendency to isolate false positives when screening with Geneticin®. It
is very important to purify your putative Geneticin® resistant clones by
streaking for single colonies on YPD and then confirming Geneticin® resistance
on YPD-Geneticin® plates. For this reason, we do not recommend replica-plating
as a method to screen for Geneticin® resistance. If you do elect to replica-plate,
be sure to confirm Geneticin® resistance.
Prepare 4 YPD plates containing the following concentrations of Geneticin®: 0,
0.25, 0.5, 0.75, 1.0, 1.5, 1.75, 2.0, 3.0, and 4.0 mg/mL (see Recipes, page 36).
Continued on next page
27
pPIC3.5K–In Vivo Screening of Multiple Inserts, Continued
Method 1,
(Spheroplasts)
Use this procedure if you transformed Pichia spheroplasts. Start with plates
containing His+ transformants.
1.
Using a sterile spreader, remove the top layer of the soft agar containing the
His+ transformants and place into a sterile, 50 mL conical centrifuge tube.
2.
Add 10 to 20 mL of sterile water. There should be a 2X volume of water
above the settled agar. Vortex vigorously for 1 to 2 minutes.
3.
Set centrifuge tube upright on bench and let agar pieces settle (about 1
minute).
4.
Determine the cell density of the supernatant by using a hemacytometer.
You need at least 5 × 105 cells/mL so you can plate ~105 cells in 200 L or
less.
(If the cells are too dilute, transfer the liquid to a fresh tube and centrifuge
the cells. Resuspend the cell pellet in sterile water in a volume sufficient to
give 5 × 105 cells/mL.).
5.
Plate 105 cells on YPD-Geneticin® plates containing Geneticin® at a final
concentration of 0.25, 0.5, 0.75, 1.0, 1.5, 1.75, 2.0, 3.0, and 4.0 mg/mL. Use
four plates for each concentration.
(You may want to confirm the titer of the cells on the YPD plates without
Geneticin® to calculate the percent of Geneticin® resistant colonies you
obtain for each Geneticin® concentration and determine whether you are
getting multimers at 1–10% of the transformants plated. Prepare 10-5,
10-6, and 10-7 dilutions of the pooled transformants using sterile water. Plate
100 to 200 L per plate.).
6.
Incubate plates at 30°C and check daily. Geneticin®-resistant colonies will
take 2 to 5 days to appear while cells plated on YPD without Geneticin® will
take 2–3 days. Proceed to Analyzing the Results, page 31.
Continued on next page
28
pPIC3.5K–In Vivo Screening of Multiple Inserts, Continued
Method 1,
(Electroporation)
Use this procedure if electroporation was used to transform Pichia.
Transformants will not be plated in top agar. Start with plates containing His+
transformants.
1.
Pipette 1 to 2 mL sterile water over the His+ transformants on each plate.
Use all the plates that have His+ transformants.
2.
Resuspend the His+ transformants into the water by using a sterile spreader
and running it across the top of the agar. Be careful not to tear the agar.
3.
Transfer and pool the cell suspension into a sterile, 50 mL conical centrifuge
tube and vortex briefly (5 to 10 seconds).
4.
Determine cell density using a spectrophotometer (1 OD600 = 5 × 107
cells/mL).
Note: Any agar present will interfere with a spectrophotometer reading.
5.
Plate 105 cells on YPD plates containing Geneticin® at a final concentration
of 0.25, 0.5, 0.75, 1.0, 1.5, 1.75, 2.0, 3.0, and 4.0 mg/mL.
(You may want to confirm the titer of the cells on the YPD plates without
Geneticin® to calculate the percent of Geneticin® resistant colonies you
obtain for each Geneticin® concentration and determine whether you are
getting multimers at 1–10% of the transformants plated. Prepare 10-5,
10-6, and 10-7 dilutions of the pooled transformants using sterile water. Plate
100 to 200 L per plate.)
6.
Incubate plates at 30°C and check daily. Geneticin®-resistant colonies will
take 2 to 5 days to appear while cells plated on YPD will take 2–3 days.
Proceed to Analyzing the Results, page 31.
If you do not plate all of the cell suspension from either of the methods above,
add sterile glycerol to 15% and freeze in convenient aliquots at –80°C. You may
thaw the aliquots and analyze for Geneticin® resistant colonies at a later date.
Continued on next page
29
pPIC3.5K–In Vivo Screening of Multiple Inserts, Continued
Method 2
You will need three sets of two microtiter plates (6 total) to screen ~180 His+
recombinants. It is important to grow your clones to approximately the same cell
density by successive inoculations to ensure that equivalent numbers of cells are
spotted on Geneticin® plates. If you plated your transformants in top agar, it
may be necessary to extract them from the agarose and re-plate them on minus
histidine plates (see Pichia Expression System manual) to pick colonies.
Remember to include controls for strain background and one copy of your gene.
For every 180 colonies, you can expect to isolate 1–10 Geneticin® resistant
colonies.
1.
Using sterile technique, add 200 L YPD to each microtiter well.
2.
Inoculate each well of the first set of plates with a single His+ transformant
using a sterile toothpick and stirring to resuspend cells.
3.
Cover the microtiter plate and incubate at 30°C for 2 days (shaking not
required).
4.
After 2 days, take new microtiter plates and add 190 L of YPD to each well.
5.
Inoculate the second set of microtiter plates with 10 L from the first set of
microtiter plates by using a multichannel pipette. Make sure the second set
of plates is marked and oriented in such a way that you can keep track of
wells.
6.
Cover and incubate the second set of plates overnight at 30°C.
7.
The next day, repeat Steps 5 and 6, creating a third set of microtiter plates.
Note: Successive growth and passage of clones will bring them all to the
same cell density.
8.
After incubation, take the third set of plates and resuspend the cells in each
well by pipetting up and down with a multichannel pipette set on 100 L
volume.
9.
Spot 10 L from each well on YPD plates containing Geneticin® at a final
concentration of 0, 0.25, 0.5, 0.75, 1.0, 1.5, 1.75, 2.0, 3.0, and 4.0 mg/mL. Spot
in a regular pattern using the multi-channel pipette or a grid underneath the
plate.
10. Let the liquid soak in, then incubate plates at 30°C, and check after 2, 3, 4, or
5 days for Geneticin® resistant clones. Proceed to Analyzing the Results,
next page.
Continued on next page
30
pPIC3.5K–In Vivo Screening of Multiple Inserts, Continued
Analyzing the
Results
There may be only a few Geneticin® resistant colonies, and they may be of
different sizes, but the colony morphology should be the same. Pick all
Geneticin® resistant colonies and purify by streaking for single colonies. Be sure
to confirm the observed level of Geneticin® resistance.
You may not find colonies resistant to 2.0, 3.0, or 4.0 mg/mL Geneticin®.
"Jackpot" clones resistant to these high levels of Geneticin® are very rare. You
may have to screen thousands of His+ transformants to isolate colonies resistant
to 2–4 mg/mL Geneticin®.
Analyze for the presence of your insert by PCR (see the Pichia Expression System
manual for a protocol). PCR will only tell you if your gene is present. It will not
tell you how many copies of your gene are integrated or at which locus the
integration occurred. PCR can reasonably be done on 12–20 transformants.
Remember to include the vector only and original (one copy) construct controls
to analyze your PCR experiment.
MEND
ION
AT
RECOM
Since there is no guarantee that multiple copies will actually increase the
amount of protein expressed, you may elect to proceed directly to expression to
see if any of these colonies overexpress their protein. Be sure to include a single
copy insert as a control. Test all your Geneticin® resistant colonies for their Mut
phenotype so that you induce expression properly. Refer to the Pichia
Expression System manual for methods to express your protein.
Determining Copy
Number
Be sure to purify your clones by streaking for single colonies and making frozen,
glycerol stocks of all your Geneticin® resistant colonies. Always initiate
expression studies from frozen stocks, not old plates.
If you find that your Geneticin®-resistant His+ recombinants significantly
overexpress your protein, you may wish to quantify the copy number of your
gene. Copy number may be analyzed by Southern or quantitative dot (slot) blots
(see page 41). It is very important to include genomic DNA isolated from Pichia
recombinants transformed with pPIC3.5K alone and pPIC3.5K with a single
copy of your gene as controls to evaluate your experiment.
Continued on next page
31
pPIC3.5K–In Vivo Screening of Multiple Inserts, Continued
Troubleshooting
Since there is a tendency to isolate false positives (colonies which appear to be
Geneticin® resistant, but are not), it is very important to purify your putative
Geneticin® resistant colonies and confirm the observed level of Geneticin®
resistance before proceeding further.
The other most common problem with the in vivo method is that very few
Geneticin® resistant colonies are isolated requiring screening of more His+
transformants. Remember that you are isolating spontaneous, multiple
integration events. These occur at a frequency of 1–10% which may mean that
you need to screen thousands of His+ transformants as opposed to hundreds. In
addition, to isolate recombinants with the most copies of your gene inserted, you
must screen more His+ transformants. Successive multiple insertions are simply
more rare.
If you find that your transformation efficiency is low, try electroporation instead
of spheroplasting. This may increase the transformation efficiency and help you
isolate more His+ transformants.
32
Appendix
Vectors
Introduction
The vectors pPIC3.5K and pAO815 share many of the same features (see below).
Both are functional in Pichia strains GS115 and KM71. However, pPIC3.5K has a
more extensive multiple cloning site and contains the kanamycin gene for in vivo
screening of multiple copy inserts. It is identical to pPIC3.5 except for the
presence of the kanamycin gene. pAO815 is similar to pHIL-D2 except that it
does not contain an f1 origin.
Features
The table below describes the general features of the pPIC3.5K and pAO815
Pichia expression vectors.
Feature
Description
Benefit
5´ AOX1
An ~1,000 bp fragment containing the
AOX1 promoter
Allows methanol-inducible high level
expression in Pichia.
Targets plasmid integration to the AOX1 locus.
MCS
Multiple Cloning Site
Allows insertion of your gene into the
expression vector.
TT
Native transcription termination and
polyadenylation signal from AOX1 gene
(~260 bp)
Permits efficient transcription termination and
polyadenylation of the mRNA.
HIS4
Pichia wild-type gene coding for
histidinol dehydrogenase (~2.4 kb) and
used to complement Pichia his4 strains
Provides a selectable marker to isolate Pichia
recombinant strains.
3´ AOX1
Sequences from the AOX1 gene that are
further 3´ to the TT sequences (~650 bp)
Targets plasmid integration at the AOX1 gene.
Amp
pBR322 origin
BamH I
Bgl II
Not I
Sac I
Sal I
Stu I
Ampicillin resistance gene
E. coli origin of replication
Allows selection, replication, and maintenance
in E. coli.
Unique restriction sites
(Note: Stu I is not unique to pPIC3.5K)
Permits linearization of vector for efficient
integration into the Pichia genome and
generation of either Mut+ or MutS recombinants.
Kanamycin resistance gene from Tn903
which confers resistance to Geneticin® in
Pichia and kanamycin resistance in E. coli
(for pPIC3.5K only)
Allows in vivo screening for multicopy inserts by
increased resistance to Geneticin®.
Also allows selection for kanamycin resistance
in E. coli.
kan
There is no yeast origin of replication in any of the Pichia expression vectors
available from Invitrogen. His+ transformants can only be isolated if
recombination occurs between the plasmid and the Pichia genome (i. e.
integration of the plasmid).
33
pPIC3.5K
The figure below shows the map of pPIC3.5K. Details of the multiple cloning site
are shown on page 9.
BamH I
SnaB I
EcoR I
Avr II
Not I
Map of pPIC3.5K
Sac I
Comments for pPIC3K:
9004 nucleotides
TT
3´ AOX1 (TT)
HIS4
pPIC3.5K
Sal I
9.0 kb
'A
3
34
2
p BR 3 2
5´ AOX1 promoter fragment: bases 1-937
5´ AOX1 primer site: bases 855-875
Multiple Cloning Site: bases 938-968
3´ AOX1 primer site: bases 1055-1075
3´ AOX1 transcription
termination (TT): bases 981-1314
HIS4 ORF: bases 4242-1708
Kanamycin resistance gene: bases 5458-4656
3´ AOX1 fragment: bases 5850-6607
pBR322 origin: bases 7689-7016
Ampicillin resistance gene: bases 8694-7834
Amp
ici
llin
OX1
5' A
BspE I
OX
1
Kana m y
cin
pAO815
The figure below shows the map of pAO815. Details of the multiple cloning site
are shown on page 10.
EcoR I
Map of pAO815
3´ AOX1 (TT)
Bgl II
Comments for pAO815:
7709 nucleotides
TT
pAO815
BamH I
HIS4
p BR 3
Sal I
Stu I
7.7 kb
22
5´ AOX1 promoter fragment: bases 1-940
5´ AOX1 primer site: bases 855-875
EcoR I Site: bases 943-948
3´ AOX1 primer site: bases 1024-1044
3´ AOX1 transcription
termination (TT): bases 950-1277
HIS4 ORF: bases 4199-1665
3´ AOX1 fragment: bases 4554-5310
pBR322 origin: bases 6394-5740
Ampicillin resistance gene: bases 7399-6539
Amp
ici
llin
X1
5' AO
3' A
OX1
Bgl II
35
Recipes
YPD-Geneticin®
plates
Yeast Extract Peptone Dextrose Medium
1% yeast extract
2% peptone
2% dextrose (glucose)
1.5% agar
Variable amounts of Geneticin®
10X D (20% Dextrose)
Dissolve 200 g of D-glucose in 1,000 mL of water. Autoclave for 15 minutes or
filter sterilize. The shelf life of this solution is approximately one year.
100 mg/mL Geneticin®
Geneticin® is available from Invitrogen (see page 44)
Prepare 30 mL of 100 mg/mL Geneticin® stock solution in sterile water. Filter
sterilize and store frozen at –20°C. You will use this solution to make YPD plates
containing Geneticin® at final concentrations of 0.25, 0.5, 0.75, 1.0, 1.5, 1.75, 2.0,
3.0, and 4.0 mg/mL.
For 250 mL (8 to 10 plates of a single Geneticin® concentration):
36
1.
Combine 2.5 g yeast extract, 5 g peptone, and 5 g agar in 225 mL deionized
water.
2.
Autoclave for 20 minutes on liquid cycle.
3.
Add 25 mL of 10X D and mix well.
4.
Cool YPD to approximately 55–60°C and add appropriate volume of
Geneticin® stock (see chart below). Remember to also make several YPD
plates without Geneticin®.
5.
Mix well by swirling, but be careful to minimize bubble formation.
6.
Pour agar solution into 10 cm petri plates. Let plates harden, invert, and
store bagged at 4°C. Plates are stable for at least 6 months.
Final [Geneticin®] (mg/mL)
mL Geneticin® stock/250 mL YPD
0.25
0.625
0.50
1.25
0.75
1.875
1.00
2.5
1.50
3.75
1.75
4.375
2.00
5.0
3.00
7.5
4.00
10.0
Pichia Genomic DNA Isolation
Introduction
The protocol below allows you to isolate DNA from the desired His+
recombinant and untransformed GS115 or KM71. The DNA isolated is suitable
for Southern blot analysis, dot/slot blot analysis or genomic PCR. See Current
Protocols in Molecular Biology, pages 13.11.1 to 13.11.4 (Ausubel, et al., 1990), Guide
to Yeast Genetics and Molecular Biology, pages 322–323 (Strathern and Higgins,
1991), or Holm, et al., 1986 for other methods to isolate DNA from Pichia.
In addition to the protocol listed below, we use our Easy-DNA™ Kit (see page
44) to isolate DNA from Pichia for PCR and quantitative dot (slot) blots. See page
41 for this protocol.
Lastly, there is a fast DNA isolation protocol for multiple samples (24) which has
been reported (Wach, et al., 1994).
Solutions
Preparation
You need to prepare the following solutions.

Minimal Medium (MD, MGY)

Sterile water

Fresh, SCED (1 M sorbitol, 10 mM sodium citrate, pH 7.5, 10 mM EDTA, 10
mM DTT)

Zymolyase, 3 mg/mL stock solution in water (Seikagaku America, Inc.)

1% SDS in water

5 M potassium acetate, pH 8.9

TE buffer, pH 7.4 (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, pH 8.0)

7.5 M ammonium acetate, pH 7.5

Phenol:chloroform (1:1 v/v)
1.
Grow the recombinant strain and the parent strain at 30°C to an OD600 of 5–
10 in 10 mL of minimal media such as MD or MGY (recombinant) or MDH
or MGYH (GS115).
2.
Collect the cells by centrifugation at 1500 × g for 5–10 minutes at room
temperature.
3.
Wash the cells with 10 mL sterile water by centrifugation as in Step 2.
Spheroplasting and 1. Resuspend the cells in 2 mL of SCED buffer, pH 7.5. Make this solution
fresh.
Lysis
2.
Add 0.1–0.3 mg of Zymolyase (mix well before adding to the cells). Incubate
at 37°C for 50 minutes to achieve ~80% spheroplasting.
3.
Add 2 mL of 1% SDS, mix gently, and set on ice for 5 minutes.
4.
Add 1.5 mL of 5 M potassium acetate, pH 8.9, and mix gently.
5.
Centrifuge at 10,000 × g for 5–10 minutes at 4°C and save the supernatant.
Continued on next page
37
Pichia Genomic DNA Isolation, Continued
Precipitating DNA
38
1.
Transfer the supernatant from Step 5, previous page, and add 2 volumes of
ethanol to the supernatant. Incubate at room temperature for 15 minutes.
2.
Centrifuge at 10,000 × g for 20 minutes at 4°C.
3.
Resuspend the pellet gently in 0.7 mL TE buffer, pH 7.4 and transfer to a
microcentrifuge tube.
4.
Gently extract with an equal volume of phenol:chloroform (1:1 v/v)
followed by an equal volume of chloroform:isoamyl alcohol (24:1). Split the
aqueous layer into two microcentrifuge tubes.
5.
Add 1/2 volume of 7.5 M ammonium acetate, pH 7.5, and 2 volumes of
ethanol to each tube. Place on dry ice for 10 minutes or at –20°C for
60 minutes.
6.
Centrifuge at 10,000 × g for 20 minutes at 4°C and wash the pellets once with
1 mL of 70% ethanol. Briefly air dry the pellets and resuspend each one in
50 L of TE buffer, pH 7.5. Determine the concentration of the DNA sample.
The two samples can be stored separately or combined, and stored at –20°C
until ready for use.
Easy-DNA™ Protocol for Isolation of DNA from Pichia
Solutions
Preparing Cells
You will need to prepare the following solutions.

Minimal Medium (MD, MGY)

TE buffer, pH 7.4 (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, pH 8.0)

1 M Sorbitol, 100 mM EDTA, 14 mM -mercaptoethanol (make fresh)

Zymolyase, 3 mg/mL stock solution in water (Seikagaku America, Inc.)

SCED (1 M sorbitol, 10 mM sodium citrate, pH 7.5, 10 mM EDTA, 10 mM
DTT, make fresh)

Easy-DNA™ Kit (to purchase, see page 44)

Chloroform

Isopropanol

70% or 80% ethanol

RNase A
1.
Grow the recombinant strain and the parent strain at 30°C to an OD600 of
5-10 in 2–5 mL of minimal media such as MD or MGY (recombinant) or
MDH or MGYH (GS115 or KM71).
2.
Harvest 1.5 mL of the culture by centrifuging at maximum speed in a
microcentrifuge for 1–2 minutes at room temperature.
3.
Resuspend cells in 1.5 mL TE and centrifuge as in Step 2.
4.
Resuspend cells in 1 mL fresh 1 M Sorbitol, 100 mM EDTA, 14 mM
-mercaptoethanol. Vortex to resuspend.
5.
Add 1.5 L of 3 mg/mL Zymolyase to each tube of cells and incubate at
30°C for 1 hour.
6.
Centrifuge at 1,700 × g in a microcentrifuge for 8 minutes at room
temperature. It is important to centrifuge with less force as the cells are
fragile because of digestion with Zymolyase.
7.
Gently resuspend the cells in 200 L fresh SCED and incubate at 37°C for
1 hour.
Continued on next page
39
Easy-DNA™ Protocol for Isolation of DNA from Pichia,
Continued
Isolating DNA
40
1.
Add 350 L Easy-DNA™ Solution A to the cell suspension from Step 7,
above, vortex, and incubate at 65°C for 10 minutes.
2.
Add 150 L of Easy-DNA™ Solution B and vortex.
3.
Add 600 L chloroform and vortex.
4.
Centrifuge at maximum speed for 20 minutes at room temperature.
5.
Transfer the aqueous layer to a fresh tube, add 600 L isopropanol, and mix
by inversion. Incubate at room temperature for 10 minutes.
6.
Centrifuge sample at maximum speed for 20 minutes at 4°C.
7.
Wash pellet with cold 70 or 80% ethanol, centrifuge at maximum speed for
2 minutes at 4°C, remove ethanol, and air-dry.
8.
Resuspend the pellet in 50 L TE containing 50 g/mL RNase A and
incubate overnight at room temperature. Quantify the amount of DNA. We
generally use 5 L of this DNA solution in a 50 L PCR reaction.
Determining Copy Number of Multiple Integrants
General Guidelines •
Use standard procedures and solutions for Southern blotting as outlined in
Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989), pages 9.31–
9.58.

Isolate genomic DNA and quantify using fluorometry. Be sure to eliminate
RNA. It is very important to load the same amount of DNA into each lane to
accurately determine copy number.

Probe your Southern blot with probes to both HIS4 and your gene. Note that
the point mutation in the his4 gene in the host strain will not interfere with
hybridization if you make the probe complementary to the wild-type gene.

If you used pPIC3.5K to generate multimers, use Bgl II to digest your DNA
(Clare, et al., 1991a). Note that if you used pPIC3.5K that all multimers are
NOT necessarily in a head-to-tail configuration. Some multimers may be
head-to-head and others tail-to-tail. We recommend that you think about
what products may be produced. An expression cassette in the opposite
orientation may produce a different band. The number of multiple copies
will cause one or two bands (depending on orientation) in the Southern blot
to increase in intensity once you are >2 copies.

If you used pAO815 to generate multimers, use Bgl II and BamH I to digest
the genomic DNA and release the multimer. The molecular weight of the
band should allow you to determine the number of multimers. If this
multimer is too large, you may wish to digest with an enzyme like Sac I.
This will collapse the multimer into single fragments containing your gene.
These will produce a band that will be quite intense. The relative intensity of
this band versus a band containing a single copy of your gene will allow you
to determine the copy number.

Bgl II digested DNA from GS115 and GS115 transformed with pPIC3.5K or
pAO815 will produce a bands of 2.8 kb (the genomic copy of HIS4), and
~6.7 kb (the vector derived copy of HIS4), respectively, when probed with a
complementary fragment to HIS4.
41
Determining Copy Number of Multiple Integrants, Continued
Introduction
You may wish to determine the actual number of gene copies in your Pichia
recombinant. You may use quantitative dot blots or Southern hybridization to
analyze gene copy number (Brierley, et al., 1994; Clare, et al., 1991a; Romanos, et
al., 1991; Scorer, et al., 1993; Scorer, et al., 1994). This requires isolation of genomic
DNA from Pichia recombinants transformed with the parent vector (0 copies of
your gene), pAO815 or pPIC3.5K containing 1 copy of your gene (single copy
control), and the Pichia recombinants containing multiple copies of your gene.
Use the protocols detailed on the pages 37 and 39 to isolate genomic DNA.
Quantitative Dot
Blot Solutions
You will need the following solutions, 10–15 mL of each for each dot blot.
Quantitative Dot
Blot Procedure

50 mM EDTA, 2.5% -mercaptoethanol pH 9.0

1 mg/mL Zymolyase 100T in water (Seikagaku America, Inc.)

0.1 N NaOH, 1.5 M NaCl, 0.015 M sodium citrate

2X SSC (1X = 0.15 M NaCl, 0.015 M sodium citrate)

3MM paper
The following protocol is a summary of a rapid DNA dot blot technique to
detect multiple integrants (Romanos, et al., 1991). It is very important to spot
equivalent numbers of cells onto filters to quantify copy number.
1.
Grow Mut+ or Muts transformants in individual wells of a 96-well microtiter
plate in 200 L of YPD broth at 30°C until all wells have approximately the
same density. This may necessitate several passages; see page 27 for more
details. Alternatively, individual transformants may be grown in culture
tubes and the absorbance at 600 nm normalized with the addition of
medium.
2.
Filter 50 L of each sample onto a nitrocellulose or nylon filter placed into a
dot (slot) blot apparatus using multichannel pipettor. Air dry filters.
3.
To lyse the cells on the filter, treat the filter with four solutions as follows:
place two sheets of 3MM paper in a tray and soak with 10–15 mL of 50 mM
EDTA, 2.5% -mercaptoethanol pH 9.0. Make sure that the paper is
uniformly soaked and that there are no puddles. Place the nitrocellulose
filter face down on the treated 3MM paper. Incubate for 15 minutes at room
temperature.
4.
Remove the nitrocellulose filter from the 3MM paper and replace the 3MM
paper with two new sheets. Soak the paper with 10–15 mL of 1 mg/mL
Zymolyase 100T as described in Step 3. Place the nitrocellulose filter face
down on the 3MM paper and incubate for 4 hours at 37°C.
5.
Remove the nitrocellulose filter from the paper and replace the paper with
two new sheets. Soak the paper with 10–15 mL of 0.1 N NaOH, 1.5 M NaCl,
0.015 M sodium citrate. Place the nitrocellulose filter face down on the paper
and incubate for 5 minutes at room temperature.
Continued on next page
42
Determining Copy Number of Multiple Integrants, Continued
Quantitative Dot
Blot Procedure,
Continued
6.
Remove the nitrocellulose filter and replace with two new 3MM sheets. Soak
with 10–15 mL 2X SSC. Place the nitrocellulose filter face down on the 3MM
paper and incubate for 5 minutes at room temperature. Repeat.
7.
Bake nitrocellulose filters at 80°C or UV-crosslink DNA to nylon. The filters
may be probed with a nonradioactive-labeled or random-primed,
32P-labeled probe complementary to your gene.
Multi-copy integrants can be identified by a strong hybridization signal relative
to the single copy control. Dot blots can then be quantified for copy number by
densitometry of the film or blot, or by using a -scanner (if radiolabeled).
Southern Blot
Analysis
For a detailed description of this technique as applied to Pichia pastoris, see
(Clare, et al., 1991a). It is very important to digest your DNA with the right
restriction enzyme(s) to generate a blot of digested and gel-separated genomic
DNA. It is also important to understand that your strategy will be different if
you use pPIC3.5K versus pAO815 to generate your multiple copies. Digestion of
DNA from Pichia recombinants containing multiple copies will produce a band
that will vary in intensity depending on the number of copies of your gene. It is
very important to include a control to show the intensity of a single copy gene.
The band intensities can be relatively quantified using densitometry to estimate
gene dosage.
Controls
It is very important to include DNA from the host strain alone (GS115 or KM71),
the host strain transformed with the parent vector (pPIC3.5K or pAO815), and
the host strain transformed with a vector containing one copy of your gene. It is
also a very good idea to make a probe to the HIS4 gene as an internal control for
single copy in addition to a probe to your gene. Note that if your gene inserts
into his4, two copies of the HIS4 gene are created, one mutant and the other
wild-type (see Recombination and Integration in Pichia, Pichia Expression Kit
manual).
43
Accessory Products
Introduction
The following products may be used with the pPIC3.5K and pAO815 vectors. For
details, visit www.invitrogen.com or contact Technical Support (see page 45).
Item
Amount
Catalog no.
Electrocomp TOP10F´
5 × 80 L
C665-55
One Shot® TOP10F´ (Chemically
Competent cells)
20 × 50 L
C3030-03
25 preps
K1999-25
1 gram
11811-023
5 grams
11811-031
25 grams
11811-098
™
S.N.A.P.™ Gel Purification Kit
®
Geneticin
Primers
For your convenience, Invitrogen offers a custom primer synthesis service. Visit
www.invitrogen.com for more details.
Other Pichia
Products
Other Pichia products available from Invitrogen are described below:
Item
Amount
Catalog no.
Pichia Expression Kit
Complete Kit for Gene Expression in Pichia
pastoris.
1 kit
K1710-01
Spheroplast Kit
Preparation of Pichia spheroplasts
1 kit
K1720-01
Pichia
Transformation Kit
Rapid preparation and transformation of
competent P. pastoris cells.
1 kit
K1730-01
Easy-DNA™ Kit
Isolation of DNA from Pichia for PCR.
1 kit
K1800-01
EasyComp™
44
Purpose
pPICZ A, B, & C
and
For simple selection on
intracellular expression of recombinant
proteins containing a C-terminal histidine tag.
20 g each
V190-20
pPICZ A, B, &C
For simple selection on Zeocin™ and secreted
expression of recombinant proteins containing
a C-terminal histidine tag.
20 g each
V195-20
pPIC9K
For in vivo isolation of multiple copy inserts
for secreted expression.
20 g
V175-20
Zeocin™
Technical Support
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
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
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45
Purchaser Notification
Limited Use Label
License No. 74:
Pichia Pastoris
Expression
System
46
The Pichia Expression System is based on the yeast Pichia pastoris. Pichia pastoris was developed into an
expression system by scientists at Salk Institute Biotechnology/ Industry Associates (SIBIA) and Phillips
Petroleum for high-level expression of recombinant proteins. All patents for Pichia pastoris and licenses
for its use as an expression system are owned by Research Corporation Technologies (RCT), Inc., Tucson,
Arizona. Life Technologies has an exclusive license to sell Pichia expression kits and vectors to scientists
for research purposes only, under the terms described below. Use of Pichia pastoris by commercial
entities for any commercial purpose requires the user to obtain a commercial license as detailed below.
Before using any Pichia expression product, please read the following license agreement. If you do not
agree to be bound by its terms, contact Life Technologies within 10 days for authorization to return the
unused Pichia expression products and to receive a full refund. If you do agree to the terms of this license
agreement, please complete the User Registration Card and return it to Life Technologies before using
the product.
Life Technologies Corporation ("Life Technologies") grants you a non-exclusive license to use the
enclosed Pichia expression vectors ("Expression Vector") for academic research or for evaluation
purposes only. The Expression Vectors are being transferred to you in furtherance of, and reliance on,
such license. You may not use the Expression Vectors for any commercial purpose without a license for
such purpose from Research Corporation Technologies, Inc., Tucson, Arizona.
Commercial purposes include: any use of Expression Products or Expression Vectors in a Commercial
Product; any use of Expression Products or Expression Vectors in the manufacture of a Commercial
Product; any sale of Expression Products; any use of Expression Products or the Expression Kit to
facilitate or advance research or development directed to a Commercial Product; and any use of
Expression Products or the Expression Kit to facilitate or advance any research or development program
the results of which will be directly applied to the development or manufacture of a Commercial
Product. "Expression Products" means products expressed with the Expression Kit, or with the use of
any Pichia expression vectors (including the Expression Vector) or host strains. "Commercial Product"
means any product intended for sale or commercial use.
Commercial entities may conduct their evaluation for one year at which time this license automatically
terminates. Commercial entities will be contacted by Research Corporation Technologies during the
evaluation period regarding their desire for a commercial license.
Access to the Expression Kit and Vector must be limited solely to those officers, employees and students
of your institution who need access to perform the above-described research or evaluation. You must
inform each such officer, employee and student of the provisions of this license agreement and require
them to agree, in writing, to be bound by the provisions of this license agreement. You may not
distribute any Expression Vector or host strain contained herein or in the Expression Kit to others, even
those within your own institution. You may only transfer modified, altered, or original material from the
Expression Kit or Vector to a third party following written notification of, and written approval from,
Life Technologies so that the recipient can be licensed. You may not assign, sub-license, rent, lease or
otherwise transfer this license agreement or any of the rights or obligation there under, except as
expressly permitted by Life Technologies and RCT.
This license agreement is effective until terminated. You may terminate it at any time by destroying all
Pichia Expression products in your control. It will also terminate automatically if you fail to comply with
the terms and conditions of the license agreement. You shall, upon termination of the license
agreement, destroy all Pichia Expression products in your control, and so notify Life Technologies in
writing.
You may contact Research Corporation Technologies at the following address: Bennett Cohen, Ph.D.,
Research Corporation Technologies, 101 North Wilmot Road, Suite 600, Tucson, Arizona 85711-3335. Tel:
520-748-4443, Fax: 520-748-0025.
References
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Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, New
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Cavener, D. R. and Stuart, C. R. (1991) Eukaryotic Start and Stop Translation Sites. Nucleic Acids Res. 19:
3185-3192.
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Expression of Tetanus Toxin Fragment c in Pichia pastoris Strains Containing Multiple Tandem
Integrations of the Gene. Bio/Technology 9: 455-460.
Clare, J. J., Romanos, M. A., Rayment, F. B., Rowedder, J. E., Smith, M. A., Payne, M. M., Sreekrishna, K.
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Fairweather, N. F. and Charles, I. G. (1991) Recombinant Bordetella pertussis Pertactin p69 from the Yeast
Pichia pastoris High Level Production and Immunological Properties. Vaccine 9: 901-906.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring
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Scorer, C. A., Buckholz, R. G., Clare, J. J. and Romanos, M. A. (1993) The Intracellular Production and
Secretion of HIV-1 Envelope Protein in the Methylotrophic Yeast Pichia pastoris. Gene 136: 111-119.
Scorer, C. A., Clare, J. J., McCombie, W. R., Romanos, M. A. and Sreekrishna, K. (1994) Rapid Selection
Using Geneticin® of High Copy Number Transformants of Pichia pastoris for High-level Foreign Gene
Expression. Bio/Technology 12: 181-184.
Strathern, J. N. and Higgins, D. R. (1991) Recovery of Plasmids from Yeast into Escherichia coli: Shuttle
Vectors. In: Guide to Yeast Genetics and Molecular Biology (C. Guthrie and G. R. Fink, eds), Methods in
Enzymology, (J. N. Abelson and M. I. Simon, eds). Volume 194. Academic Press, San Diego, CA.
Thill, G. P., Davis, G. R., Stillman, C., Holtz, G., Brierley, R., Engel, M., Buckholz, R., Kinney, J., Provow, S.,
Vedvick, T. and Siegel, R. S. (1990) Positive and Negative Effects of Multi-Copy Integrated Expression
in Pichia pastoris. International Symposium on the Genetics of Microorganisms, 2: 477-490.
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High-level Secretion of Biologically Active Aprotonin from the Yeast Pichia pastoris. J. Ind. Microbiol. 7:
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Wach, A., Pick, H. and Phillippsen, P. (1994) Procedures for Isolating Yeast DNA for Different Purposes:
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