Thermo Fisher Scientific 1-Step Human High-Yield Maxi IVT Kit User Guide

INSTRUCTIONS
1-Step Human High-Yield
Maxi IVT Kit
88892
2569.0
Number
Description
88892
1-Step Human High-Yield Maxi IVT Kit, contains sufficient reagents to perform 2 reactions
(2000µL each)
Kit Contents
HeLa Lysate
Accessory Proteins
Reaction Mix
5X Dialysis Buffer
Positive Control DNA: pCFE-GFP
(0.5µg/µL, 10µg)
pT7CFE1-NHis-GST-CHA Expression Vector
(0.5µg/µL, 10µg)
Kit Contents
Maxi Dialysis Device
Nuclease-free Water
Cap Color
Red
Green
Yellow
Clear
Solid white
Clear
88892X
4 × 500µL
4 × 100µL
4 × 200µL
1 × 23mL
20µL
20µL
88892Y
2 each
2 × 50mL
Note: Completely read the instructions before proceeding with the protocols.
Storage: Upon receipt store 88892X at -80°C and 88892Y at room temperature. 88892X is shipped
with dry ice. 88892Y is shipped at ambient temperature.
Table of Contents
Introduction ................................................................................................................................................................................. 2
Procedure Summary..................................................................................................................................................................... 2
Important Product Information .................................................................................................................................................... 2
Additional Materials Required ..................................................................................................................................................... 3
Protocol for using the 1-Step Human High-Yield IVT Maxi Kit ................................................................................................ 3
A.
Protein Expression ......................................................................................................................................................... 3
B.
Determination of Protein Expression Level .................................................................................................................. 4
C.
Purification of IVT-expressed Proteins ......................................................................................................................... 4
Troubleshooting ........................................................................................................................................................................... 5
Additional Information ................................................................................................................................................................ 6
A.
pT7CFE1-NHis-GST-CHA Vector Cloning Sites and Sequence Features ................................................................... 6
B.
Vector DNA Clean-up and Concentration Protocol ...................................................................................................... 6
C.
Expression-ready Clones for Use with the 1-Step Human High-Yield IVT Kits .......................................................... 7
Related Thermo Scientific Products ............................................................................................................................................ 8
General References ...................................................................................................................................................................... 8
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Introduction
The Thermo Scientific™ 1-Step Human High-Yield Maxi IVT Kit is a mammalian in vitro translation (IVT) system based on
HeLa cell lysates, which contain all of the cellular components required for protein synthesis, including ribosomes, initiation
factors, elongation factors and tRNA. When supplemented with the included proprietary Accessory Proteins, Reaction Mix
and a DNA template cloned into the Thermo Scientific™ pT7CFE1-NHis-GST-CHA Vector, this system can synthesize
protein for up to 16 hours.
The benefits of in vitro protein expression over traditional in vivo systems include the ability to express toxic proteins, faster
protein synthesis and protein labeling with modified amino acids. The optimized kit contains a T7 promoter and an EMCV
internal ribosome entry site (IRES) to facilitate high levels of in vitro protein expression in a cap-independent fashion. Using
a vector containing the EMCV IRES element is critical for obtaining high expression levels in this human in vitro protein
expression system.
Procedure Summary
Important Product Information
•
Use the included Thermo Scientific pT7CFE1-NHis-GST-CHA Vector (Product No. 88871) for cloning and
expressing the target gene. See the Additional Information Section for additional vector choices, cloning sites and
expression-ready clones.
•
Thaw HeLa Lysate on ice, aliquot and quickly store at -80°C. All components of the kit are stable for up to five freezethaw cycles as long as the contents are stored at -80°C immediately after use. For faster thawing, gently flick the lysate
tubes.
•
Undiluted lysate and reactions containing lysate will appear cloudy before and after incubation. Accessory Proteins and
Reaction Mix may also appear clear to cloudy upon thawing; mix thoroughly but gently before and after adding each
component to the IVT reaction. Undiluted 5X Dialysis Buffer may appear cloudy; mix well before and after dispensing.
•
Avoid RNase contamination by wearing gloves; working in a clean, dust-free environment; and using RNase-free tips
and microcentrifuge tubes.
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Additional Materials Required
•
DNA preparation kit (e.g., Thermo Scientific™ GeneJET™ Plasmid Maxi Prep Kit, Product No. K0492)
•
Western immunoblot accessories for detecting expressed protein
•
FITC filter-containing device to observe the expression of GFP in positive control reactions
•
1.5mL and 15mL RNase-free microcentrifuge tubes for assembling reactions
•
RNase-free pipette tips
•
Shaker incubator capable of maintaining temperature at 30°C.
Protocol for using the 1-Step Human High-Yield IVT Maxi Kit
A. Protein Expression
1.
With the exception of 5X Dialysis Buffer, thaw all other reagents in the kit contents of 88892X and maintain on ice.
Thaw 5X Dialysis Buffer at 25-30°C for a maximum of 30 minutes, and after making a 1X mixture, maintain the diluted
buffer at 30°C.
Note: Store any unused 88892X kit components at -80°C.
Note: The 5X Dialysis Buffer may appear cloudy. Mix or vortex gently. Do not centrifuge before use. Once diluted, the
1X Dialysis Buffer will become clear within minutes.
2.
Combine 5X Dialysis Buffer and Nuclease-free Water (volumes per Table 1) in the provided conical tube.
Table 1. Reconstitution of the Dialysis Buffer.
Component
5X Dialysis Buffer
Nuclease-free Water
Total
mL
8.0
32.0
40.0
3.
Place a dialysis device inside the 50mL tube containing 1X Dialysis Buffer as shown in the Procedure Summary Section.
4.
Optional: Set up a small reaction to test the integrity of the HeLa Lysate, Accessory Proteins and Reaction Mix. Add
12.5μL of HeLa Lysate, 2.5μL of Accessory Proteins, 5μL of Reaction Mix, 3μL of Nuclease-free Water and 2μL of
pCFE-GFP DNA plasmid to a nuclease-free 1.5mL microcentrifuge tube. Incubate at 30°C for 4-5 hours. See Section B,
Step 1: Quick visual detection for detecting the expressed GFP protein.
5.
Prepare IVT reactions using Table 2. Add the reagents in the order listed into a 15mL RNase/DNase-free tube. Gently
mix the reaction after each reagent addition. Incubate HeLa Lysate with Accessory Proteins for 10 minutes at room
temperature prior to adding the rest of the components.
Table 2. Components of the IVT reaction.
µL
Component
HeLa Lysate
Accessory Proteins
Reaction Mix
Cloned DNA(0.5µg/µL)
Nuclease Free water
1000
200
400
160
240
Total
2000
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6.
Briefly centrifuge the reaction mix at 10,000 × g for 2 minutes. A small pellet will be visible after centrifugation.
7.
Transfer the supernatant into the empty dialysis device and screw the cap on the device as described in the Procedure
Summary Section.
8.
Place the entire dialysis device into the 50mL tube containing Dialysis Buffer and close the screw cap.
9.
Incubate the reaction for 6-16 hours at 30°C in a shaker incubator. Shake the entire unit in a shaker incubator using the
following guidelines to determine the speed of shaking.
Shaker orbital radius
Recommended RPM
3mm (Eppendorf™ ThermoMixer™)
350
3/4 inch (New Brunswick C24)
250
1 inch (Thermo Scientific™ MaxQ™ 8000)
150
Note: Although protein expression is complete within six hours for most proteins tested, incubating up to 16 hours may
increase expression of some proteins. Optimal time to express each protein must be determined empirically. A small
white precipitate may be visible, which can be easily removed by centrifugation in the next step.
10. At the end of incubation, collect the contents from the dialysis device equally into two separate 1.5mL microcentrifiuge
tubes and centrifuge at 10,000 × g for 2 minutes prior to storage.
Note: Resulting reactions may be stored on ice for same-day use. For long-term storage, transfer the reaction contents
from the dialysis device and store separately at -20°C or colder.
11. Proteins expressed using this kit may be purified using the purification guidelines provided in the Product Blog article
“Choosing a vector and purification method for in vitro protein expression” on our website at: thermoscientific.com/pierce.
B. Determination of Protein Expression Level
Note: The GFP control protein is from the copepod Pontellina plumata. This GFP is not reactive to antibodies generated
against Aequorea victoria GFP (i.e., EGFP or other EGFP mutants). Use polyclonal antibodies to TurboGFP (Product No.
PA5-22688).
1.
Visualize or quantitate the GFP control protein using one of the following methods:
Quick visual detection: Place the GFP reaction tubes directly under a microscope or imaging equipment containing a
FITC filter (ex/em: 482/502nm); alternatively, spot a small volume (1-2µL) on a piece of plastic wrap or laboratory film
and visualize with fluorescent imaging equipment.
Fluorescent plate reader: Place sample directly into a white or black 96- or 384-well plate. Evaluate signal using a
fluorescent plate reader at ex/em: 482/502nm. To quantitate GFP, compare the fluorescence to a recombinant GFP
standard curve.
2.
Visualize or quantitate non-fluorescent protein expression using one of the following methods:
Fast Western immunoblot analysis: This is a quick protocol consisting of transfer and detection of proteins separated
on SDS-PAGE using ultra-sensitive Thermo Scientific™ SuperSignal™ Substrate. A detailed protocol and reagents
required for Western blot detection can be found at thermoscientific.com/pierce; search using “fast western blot.”
SDS-PAGE analysis: Separate proteins by SDS-PAGE and stain using Thermo Scientific™ GelCode™ Blue Stain
Reagent (Product No. 24590), Thermo Scientific™ Imperial™ Protein Stain (Product No. 24615) or Thermo
Scientific™ PageBlue™ Protein Staining Solution (Product No. 24620) (Figure 2).
C. Purification of IVT-expressed Proteins
Proteins expressed using this kit may be purified using the purification guidelines provided in the Product Blog article
“Choosing a vector and purification method for in vitro protein expression” on our website at: thermoscientific.com/pierce.
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Troubleshooting
Problem
Possible Cause
Solution
GFP not detected by
fluorescence in positive
control reaction
Incorrect filter set was used
The excitation/emission wavelengths of GFP are
482/502nm
Lysates have become inactive
Store unused lysate in nuclease-free tubes at -80°C; do
not exceed five cycles of freezing and thawing
No expression of target
protein
Incorrect vector was used
Use cloning vector pT7CFE1-NHis-GST-CHA provided
in the kit to clone and express the gene of interest
Note: The 1-Step Human IVT Kits are optimized using
the pCFE1 vector and its derivatives; for a complete
listing, please visit thermoscientific.com/pierce
HeLa Lysate, Accessory Proteins
and Reaction Mix were stored at a
suboptimal temperature
Store unused HeLa Lysate, Accessory Proteins and
Reaction Mix in nuclease-free tubes at -80°C; do not
exceed five cycles of freezing and thawing
Poor-quality DNA
Ethanol precipitate the DNA to remove trace amounts of
inhibitors or salts − see the Additional Information
Section for the recommended protocol
Degradation of mRNA in the
translation reaction
Maintain an RNase-free environment by wearing
gloves; working in a clean, dust-free environment; and
using RNase-free tips and microcentrifuge tubes
Protein was sensitive to proteases
Add Thermo Scientific™ Halt™ Protease Inhibitor
Single-Use Cocktail, EDTA-free (100X) (Product No.
78425) at 0.5X to the reaction mix in Step A, 5, Table 2
Low yield of target
proteins
Incorrect incubation temperature
Perform reactions at 30°C
Incorrect order of reagent addition
Incubate HeLa Lysate with Accessory Proteins for
5-10 minutes before adding remaining components to
improve target protein expression
Smaller band size than
predicted
Stop codons were in genes of
interest
Ensure the cloned genes do not have a stop codon in the
open reading frame
Protein appears to be
degraded
Proteins were susceptible to
proteases
Add Halt Protease Inhibitor Single-Use Cocktail,
EDTA-free (100X) (Product No. 78425) at 0.5X to the
reaction mix in Step A, 5, Table 2
Larger band size than
predicted
Post-translation modifications
HeLa Lysate is capable of protein post-translational
modifications, including partial glycosylation and
phosphorylation. Validate the presence of glycosylation
by digesting a small portion of the sample with Endo H
or PNGase (a loss of the higher molecular weight bands
indicates proteins were glycosylated)
Low protein yield after
purification
Reaction scale was too small
Follow guidelines provided in the Product Blog article
“Choosing a vector and purification method for in vitro
protein expression” on our website at:
thermoscientific.com/pierce
Increase reaction size
Low protein yield after
purification
Affinity tag was not accessible
Use different affinity purification for the tagged protein
Purify protein under denaturing conditions (e.g., 8M
urea) using the Thermo Scientific™ HisPur™ Cobalt
Purification Kit (Product No. 90090)
Reduce incubation temperature to 25°C
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Additional Information
A. pT7CFE1-NHis-GST-CHA Vector Cloning Sites and Sequence Features
The 1-Step High-Yield IVT Kit has been optimized using the pT7CFE1-NHis-GST-CHA cloning vector, which is designed
for high-level protein expression. In addition to multiple purification tags, it contains an HRV 3C cleavage site for tag
removal. For a complete listing of pT7CFE1 expression vector derivatives, visit thermoscientific.com/pierce; search using
“expression vectors.”
Features:
•
10 unique restriction sites are provided in the multiple cloning site for cloning genes of interest (Figure 1)
•
5´ UTR consisting of EMCV internal ribosome entry site (IRES) required for high-level protein expression
•
Poly A sequence in the 3´ region promotes mRNA stabilization and protection from nucleases
•
T7 terminator ensures synthesis of accurate sized mRNA transcripts
Figure 1. The Thermo Scientific pT7CFE1-NHis-GST-CHA Vector multiple cloning site, with the exception of Msc 1, is
common to all of the expression vectors used in the Thermo Scientific 1-Step Human IVT Kits. The translational start site is
the ATG found upstream of the His tag region.
B. Vector DNA Clean-up and Concentration Protocol
Prepare DNA using a standard maxi- or mini-prep protocol. To avoid compromising protein expression yield, completely
remove contaminating proteins and eliminate the RNase A used in many mini-prep protocols. Perform the following steps to
precipitate and, subsequently, concentrate the DNA.
1.
Add 1/10 volume of 3M sodium acetate, pH 5.5 and two volumes of ethanol. Thoroughly mix the reaction and incubate
at -20°C for 15 minutes.
2.
Centrifuge the mixture at 14,000 × g for 15 minutes. Remove the supernatant and wash the pellet once with 70% ethanol.
3.
Centrifuge at 14,000 × g for 5 minutes. Using a fine tip, remove all of the supernatant, including the residual. Air-dry the
pellet for 5 minutes at room temperature.
4.
Resuspend the pellet in nuclease-free water before measuring the DNA concentration. DNA templates may be stored in a
Tris-based buffer. It is not necessary to linearize the plasmid DNA before use.
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C. Expression-ready Clones for Use with the 1-Step Human High-Yield IVT Kits
•
Custom cloning service; please visit thermoscientific.com/pierce and search for “cloning service.”
•
The pANT7 vector library from the ASU Biodesign Institute DNASU Plasmid Repository is compatible with our 1-Step
High Yield IVT Kit. Visit http://dnasu.asu.edu/DNASU/Home.jsp for information and ordering. Under advanced search
options choose “pANT7” for vector selection.
•
PCR templates: see Tech Tip #72: PCR protocol for generating optimized templates for Pierce™ Human In Vitro
Expression Kits on our website.
Figure 2. Expression of coomassie-stainable proteins using the Thermo Scientific 1-Step Human High-Yield IVT
Kit. Five expression-ready clones (pANT7 vector) obtained from the DNASU Plasmid Repository were used to express
the GST-fusion proteins listed in Lanes 3-7. Lane 2 shows expression of the control pCFE-GFP plasmid. Reaction
mixtures of 5µL were separated by SDS-PAGE and stained with GelCode Blue Stain Reagent. Arrows indicate the
positions of expressed proteins.
Figure 3. Purification of N-terminal GST fusion proteins with immobilized glutathione. Purification of either GST-fused
proteins or untagged protein was performed as described with 10mM glutathione or HRV3C protease, respectively, using
instructions provided in the Product Blog article “Choosing a vector and purification method for in vitro protein expression”
on our website at: thermoscientific.com/pierce. The additional bands denoted with a * found with the purification of Bad are
14-3-3 proteins which co-elute with Bad. Protein identification was verified by mass spectrometry (data not shown).
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Related Thermo Scientific Products
88859-71
pT7CFE1-based Expression Vectors
88899
Recombinant GFP Protein
88881-2
1-Step Human Coupled IVT Kit – DNA
88890-1
1-Step High Yield Mini IVT Kit
MA121315
Mouse anti-6x-His Epitope Tag Monoclonal Antibody (HIS.H8)
26183
Mouse anti-HA Monoclonal Antibody (2-2.2.14)
MA4004
Mouse anti-Glutathione S-transferase Monoclonal Antibody (8-326)
35035
Pierce Fast Semi-Dry Transfer Buffer (10X), 500mL
88217
Pierce Fast Semi-Dry Blotter
35050
Pierce Fast Western Blot Kit, ECL Substrate
88221
HisPur Ni-NTA Resin, see our website for all related products
89964
HisPur Cobalt Resin, see our website for all related products
16100
Pierce Glutathione Agarose, see our website for all related products
26182
Pierce Anti-HA Agarose, see our website for all related products
K0492
GeneJET Plasmid Maxiprep Kit, see thermoscientific.com/onebio
PA5-22688
Anti-TurboGFP Polyclonal Antibody
88836-7
Pierce Anti-HA Magnetic Beads
88821-2
Pierce Glutathione Magnetic Beads
88831-2
HisPur Ni-NTA Magnetic Beads
General References
Imataka, H., et al. (2009). Advantages of human cell-derived, cell-free protein synthesis systems (Japanese). Seikagaku 81(4):303-7.
Kobayashi, T., et al. (2007). An improved cell-free system for picornavirus synthesis. J Virol Methods 142(1-2):182-8.
Kozak, M. (1983). Comparison of initiation of protein synthesis in prokaryotes, eukaryotes and organelles. Microbiol Rev 47(1):1-45.
Kozak, M. (2005). Regulation of translation via mRNA structure in prokaryotes and eukaryotes. Gene 361:13-37.
Mikami, S., et al. (2006). An efficient mammalian cell-free translation system supplemented with translation factors. Protein Expr Purif 46(2):348-57.
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