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Overview of Human in vitro Translation Systems

Learn about the features and types of Thermo Scientific 1-Step Human IVT Products.

1-Step Human IVT Product Selection GuideThe Thermo Scientific 1-Step Human In Vitro Translation System is a method for expressing proteins from DNA or mRNA templates in a cell-free solution containing essential components of the cellular translational machinery. Extracts of an immortalized human cell line provide the ribosomes, initiation and elongation factors, tRNAs and other basic components required for protein synthesis. When supplemented with proprietary accessory proteins, ATP, and an energy regenerating system, these extracts sustain the synthesis of target proteins from DNA templates for up to 6 hours without the need to remove inhibitory byproducts.

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Protein Methods Library Home

 

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Human In Vitro Translation Kits and Reagents

pT7CFE1 Expression Vectors

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Cell-free in vitro Recombinant Protein Expression

Getting Started with Human in vitro Translation

Introduction and protocol summary

1-Step Human In Vitro Translation Kits

The 1-Step Human IVT Kits use human cell lysates to synthesize proteins. Compared to bacterial (E. coli) or other (e.g., insect, wheat germ and rabbit reticulocyte lysates) systems, in vitro protein expression using human cell extracts delivers functional proteins within hours. Expression can be achieved using either a DNA or mRNA templates, which can be derived from a vector or acquired through our library of gene open reading frames.

The benefits of in vitro protein expression include compatibility with microliter-scale reactions and speed compared to traditional cell-based methods (overnight for bacterial cultures or weeks for baculoviral protein preparations). This small-scale expression method makes it easy to express numerous mutant variants simultaneously in a microplate or express large quantities of a single protein for future experiments. Small-scale synthesis also helps to avoid protein aggregation into inclusion bodies, a typical problem for bacterial expression systems. Additionally, expression of proteins in vitro enables synthesis of toxic proteins that can not be produced in live cells. The protein expression system can express protein from any species and has been validated with numerous genes from both prokaryotic and eukaryotic sources.

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Human In Vitro Translation Kits and Reagents

pT7CFE1 Expression Vectors

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Thermo Scientific cDNAs, ORFs, Promoters and other genomics-related products

Highlights:

  • Functional – uses the human translational machinery to express active proteins
  • Convenient – perform transcription and translation in a single step
  • High performance – greater yields compared to rabbit reticulocyte in vitro translation
  • Reliable – express proteins that fail in rabbit reticulocyte systems

Better Than Traditional Methods:

  • HeLa cell-free extracts are capable of expressing proteins with post-translational modifications
  • Accurate translation delivers full-length protein compatible with downstream applications
  • Protein translation is optimized with EMCV IRES element and other mRNA stabilizing elements

 

Schematic of the 1-Step Coupled Human IVT Kit for DNA
Protocol summary for the 1-Step Human Coupled IVT Kit. Simply add the appropriate template to a mixture of HeLa cell lysate, Accessory Proteins, Reaction Mix and incubate at 30°C for 90 minutes for protein yields up to 100µg/mL. Smaller reactions are ideal for expression of mutational variants in a microplate format. The reaction volumes and times can be increased to express larger amounts of a single protein for use in several downstream applications.

 

 

Selection table of available Human IVT Kits

Available Thermo Scientific 1-Step Human IVT Kits.
Kit Type Purpose
Coupled IVT Yields up to 100µg/mL per 25µL reaction in 90 minutes; protocols available for mRNA templates and for optimizing glycoprotein expression.
High-Yield IVT Yields up to 750µg/mL per 0.1mL or 2mL reaction in 6 hours to overnight; uses dialysis devices for continuous feed; protocols available for high throughput (96-well).
Heavy Protein IVT Express proteins with 90 to 95% isotope incorporation in less than 8 hours for use in mass spectrometry as controls for sample prep loss, digestion efficiency determination or as quantification standards.

 

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1-Step Human IVT Kits

Applications and functional studies with human IVT-expressed proteins

Protein function assays for enzymatic activity and protein interactions can have several obstacles. For example, the lack of good antibodies may prevent the identification and verification of proteins in complex samples via co-immunoprecipitation and electromobility supershift assays. Low expression levels can negatively affect the ability to enrich target proteins to sufficient yields for particular assays. These problems can be overcome using in vitro-expressed proteins, but many cell-free protein expression systems lack the ability or fidelity to generate high levels of functional proteins. This problem is compounded for eukaryotic proteins where proper post-translational modifications are critical.

Video feature about the Human IVT System.

 

 

Protein Interactions and the Human IVT System

Understanding the function of a protein often requires an analysis of how it interacts with other biomolecules, such as DNA, RNA and other proteins. Protein-nucleic acid (DNA or RNA) interactions can be monitored by electrophoretic mobility shift assay (EMSA) to help elucidate the regulation of mechanisms from replication to apoptosis. Protein-protein interactions can be analyzed by co-immunoprecipitation and pull-down assays to assist in the identification of novel binding partners.

For protein interaction assays, in vitro-translated protein eliminates the need to extract protein from living cells and facilitates the ability to screen multiple protein isotypes or mutants quickly and efficiently. The following are examples of protein interaction assays performed using recombinant protein generated using the Human IVT System.


A. Protein-Protein Interactions: Co-immunoprecipitation Assays

Detection of the CDK2-cyclin E1 interaction using in vitro-expressed HA-tagged CDK2
Detection of the CDK2-cyclin E1 interaction using in vitro-expressed HA-tagged CDK2. Recombinant CDK2 was expressed using the Human IVT System, a component of which is HeLa cell lysate containing endogenous cyclin E1. In vitro translation reactions were performed for HA-tagged CDK2 or a no-DNA control, and 200μL of each reaction was used for co-immunoprecipitation with an anti-cyclin E1 primary antibody. The enriched CDK2-cyclin E1 complex was recovered and eluted. The eluate (12μL) was analyzed by Western blotting along with the in vitro translation reaction mix (9uL) to detect the HA-tagged CDK2 using an anti-HA tag antibody.

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Overview of Protein-Protein Interaction Analysis

 

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Co-Immunoprecipitation Kit

B. Protein-DNA Interactions Assays: Gel Shift Assays

Detection of the CREB-DNA interaction using in vitro-translated CREB.
Detection of the CREB-DNA interaction using in vitro-translated CREB. The DNA-binding activity of CREB was detected in PC12 cell lysate or human in vitro translation reactions (CREB and vector control) using the LightShift Chemiluminescent DNA EMSA Kit. DNA-protein reactions were prepared in 10mM Tris-HCI pH 8.0, 50mM NaCl, 5mM EDTA, 25% glycerol, 0.05% NP40, 8mM spermidine and 1mM DTT. Unlabeled oligonucleotides (4pmol) were added where stated, and the reactions were incubated for 5 minutes at room temperature. Biotin-labeled oligonucleotide probe was then added, and the reactions were incubated for another 30 minutes. The complexes were then separated on a 5% TBE gel in 0.5X TBE at 100V for 2 hours at 4°C and then transferred to Biodyne B Nylon membrane (Part No. 77016) at 45V for 30 minutes (tank transfer). The complexes were then crosslinked, and the biotin was detected using the Pierce Chemiluminescent Nucleic Acid Detection Module (Part No. 89880).

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Methods for Detecting Protein-DNA Interactions

 

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LightShift Chemiluminescent EMSA Kit

C. Protein-RNA Interactions Assays: RNA Gel Shift Assays

Detect protein-RNA interactions with 1-Step Human in vitro Translation System.
Detect protein-RNA interactions with the Human IVT System. The RNA-binding ability of Aco1 was analyzed by RNA gel shift assays using in vitro-translated Aco1 prepared with the Human IVT Kit System and detected with the LightShift Chemiluminescent RNA EMSA Kit (Part No. 20158). The Aco1 translation reaction was buffer-exchanged into 1X REMSA buffer using 0.5mL Zeba Spin Desalting Columns (Part No. 89882) and pre-cleared with 30µL of High Capacity Streptavidin Agarose beads (Part No. 20361) for 30 minutes at 4°C. The pre-cleared lysate was diluted 1:20, and 2µL was incubated with 5nM biotinylated IRE-RNA in 1X REMSA buffer containing 5% glycerol. Reactions were resolved on a native 6% polyacrylamide gel in 0.5X TBE and transferred to Biodyne B Nylon membrane (Part No. 77016). Band shifts were detected using the Pierce Chemiluminescent Nucleic Acid Detection Module (Part No. 89880). Lane 1. The reaction containing only the biotinylated IRE-RNA probe (negative control). Lane 2. The band shift of the IRE-RNA probe, indicative of binding to Aco1, by the addition of in vitro-translated Aco1. Lane 3. The labeled probe is outcompeted for Aco1 binding by the addition of a 200-fold molar excess of un-labeled IRE-RNA. Lane 4. The labeled probe was not outcompeted by the addition of a 200-fold molar excess of an unlabeled non-specific RNA probe.

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Methods for Detecting Protein-RNA Interactions

 

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LightShift Chemiluminescent RNA EMSA Kit

Protein function and the Human IVT System

In vitro translation reactions are ideal for generating proteins for high-throughput mutational analysis or small-molecule screening. These assays typically require concentrated nanogram to microgram quantities of protein for analysis in multi-well plates. The Human IVT System reactions can be scaled down for 96-well (standard 25µL reaction volume) and 384-well plates (10µL reaction volume) without losing relative protein/mL yield. This feature makes it possible to perform PCR-mediated mutational analysis (see the PCR protocol) and rapidly screen libraries of protein mutants generated by in vitro translation directly in microwell plates.

For small-molecule screening, a single protein can be expressed by in vitro translation in multiple wells and then challenged against different small molecules, as demonstrated below using know protein translation inhibitors.

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1-Step Human IVT Kits

Example: caspase-3 expression and function

Expression of caspase-3 using the 1-Step Human IVT System.
Caspase-3 expressed using the human IVT system is more active than a purified protein expressed in bacteria. Equal amounts of human caspase-3 purified from E. coli or expressed with the 1-Step Human High-Yield IVT Kit were assayed for active caspase-3 activity using Caspase-glo™ Assay Reagent mix containing cleavable DEVD-aminoluciferin and luciferase substrate, according to instructions of the manufacturer (Promega Corp.).
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Expression of highly active proteins using a cell-free human IVT system

Protein screening: analyze small molecule inhibitors

Small-molecule screening using the 1-Step Human In Vitro Translation System.
Small-molecule screening using the Human IVT System. In vitro translation reactions were prepared in a 384-well microplate, and various compounds were characterized for their ability to attenuate protein synthesis. In this assay, the in vitro translation control, luciferase mRNA, expressed luciferase protein alone or in the presence of 4% dimethyl sulfoxide (DMSO), 10µg/mL cyclohexamide, 1mM staurosporine or 400µg/mL neomycin (G418). Expression was carried out for 3 hours at 30°C, and luciferase protein expression was determined by luciferase reporter assays and measured using a Thermo Scientific Varioskan Flash I instrument.

Protein types expressed

Traditional in vitro translation systems have limitations in the types of proteins that can be generated, because they based on cell-free extracts from bacteria, insect or other non-mammalian sources that require the addition of ectopic components. The Human IVT System uses human HeLa cell extract, which endogenously contains the enzymes required for protein synthesis and proper post-translational modification. Therefore, many types of proteins that can be expressed using the Human IVT Kits.

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1-Step Human IVT Kits

Express proteins across a wide range of molecular weights

Successful protein expression is influenced by protein stability, folding and solubility. High molecular weight proteins are difficult to express in vitro because of the greater chance of premature termination and the greater complexity of the tertiary structure compared to smaller proteins, which increases the chance of improper folding [1,2]. Additionally, proteins with greater size can have large hydrophobic regions that promote the formation of inclusion bodies when expressed in E. coli extracts. The 1-Step Human in vitro Translation System can be used to produce a broad range of proteins of different sizes.

Size distribution of proteins expressed by human in vitro translation
Size distribution of proteins generated with the Human in vitro Translation System. One hundred proteins ranging from 8kDa to greater than 250kDa were individually expressed using the Human in vitro Translation System with a 95% success rate.

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Cell-free In Vitro Recombinant Protein Expression

Getting Started with Human in vitro Translation

Express phosphoproteins

The 1-Step Human IVT System uses HeLa cell lysates to perform cell-free protein expression. Using a human-based system yields proteins that mirror natural proteins with respect to post-translational modifications and results in proteins that exhibit natural protein function and structure. Kinase detection by Western blot demonstrates that a protein is expressed by the Human IVT System, but detection of the total protein does not necessarily demonstrate that the protein is functional. Therefore, the phosphorylation status of three kinases (Aurora A, ERK1 and Akt), indicative of functionality, was also confirmed by Western blot using phosphor-specific antibodies. The phosphorylation status of in vitro-translated Akt was also identical in size to that in platelet-derived growth factor (PDGF)-activated NIH3T3 fibroblasts, suggesting that the Human IVT System produces functionally similar kinases to those found in vivo.

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Phosphoprotein Enrichment Kit

Phosphoprotein Phosphate Estimation Assay Kit

Thermo Scientific Pierce Antibodies

Phosphoproteins are expressed by human in vitro translation
Phosphoproteins are expressed with the 1-Step Human in vitro Translation System. Eight HA-tagged kinases were expressed using the Human IVT System and detected by Western blot analysis using an anti-HA antibody. Bands corresponding to the molecular weights of the indicated kinases were detected, indicated proper protein expression. A doublet was detected when GSK3α and GSK3β were co-expressed, and no signal was detected in the negative control (no DNA) lane.

Functional kinases are expressed by human in vitro translation
The 1-Step Human in vitro Translation System generates functional kinases. Aurora A and ERK1 were immunoprecipitated from the respective in vitro translation reactions, and phosphorylation, which is indicative of functionality, was detected by Western blot analysis using anti-phospho-Aurora A (T288) or anti-phospho-ERK 1/2 (T202/Y204) antibodies, respectively. Membranes were then stripped, and total protein levels were detected using the respective pan-specific antibodies. Non-immunoprecipitated translation reaction and a no-DNA control were concurrently analyzed. Akt phosphorylation was detected directly from the non-immunoprecipitated in vitro translation reaction and compared to endogenous Akt phosphorylation in PDGF-stimulated NIH3T3 cells. SDS-PAGE and Western blot detection of phospho-Akt and total Akt was performed as described for Aurora A and ERK1, except that anti-phospho-Akt (S473) and anti-pan Akt were used. 1, No DNA control; 2, Non-immunoprecipitated human in vitro translation reaction; 3, immunoprecipitated HA-tagged kinases; 4, PDGF-stimulated NIH3T3 cell lysate.

Express glycoproteins

The most widely used method of in vitro translation uses rabbit reticulocyte lysate [3]. While this strategy allows 5' cap-independent translation, post-translational modification of nascent proteins such as N-linked glycosylation cannot occur without adding canine microsomal membranes, which are vesicle-like remnants of the endoplasmic reticulum (ER) that form after cells are ruptured (4). Although rabbit reticulocyte lysate with microsomal membranes can generate glycosylated proteins, the approach severely reduces the total protein yield.

An adaptation of the standard protocol for the 1-Step Human Coupled IVT Kit is available, which optimizes for expression of N-linked glycosylated proteins.

Expression of N-linked glycoproteins using the 1-Step Human IVT System.
Expression of N-linked glycoprotein using Human IVT. DNAs (0.1 or 0.2μg) of HA-tagged HCGβ, ORM1 or EPO were added to 25μL coupled human IVT reactions and incubated at 26°C for 4 hours. The reactions were left untreated or treated with Endo H according to the manufacturer’s instructions and proteins separated on SDS-PAGE gel and probed for HA tag using anti-HA antibodies and western blot. The position of glycosylated (triangle markers) and unglycosylated (lowest bands) proteins are indicated. Endo H completely removes all the N-linked mannose residues resulting in the appearance of proteins as a single band. The relative proportion of glycosylated vs. unglycosylated proteins was significantly higher with lower DNA concentrations.

 

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Glycoprotein expression in a human IVT system

Express membrane proteins

Membrane proteins are often the most difficult to purify, because their hydrophobic transmembrane domains can cause large amounts of in vitro-translated protein to aggregate and precipitate. Using the Human in vitro Translation System, though, membrane proteins with varying numbers of transmembrane domains can be synthesized, including the glycophorin GypB/E, the outer mitochondrial membrane protein Bcl2-like protein Bcl2L1 and the chemokine receptor CXCR4 . Additionally, most if not all of the protein was located in the soluble fraction instead of the pellet, demonstrating that the membrane proteins generated do not aggregate and precipitate out of solution.

It is thought that the slower rate of amino acid addition and membrane-enriched HeLa extracts used in the 1-Step Human in vitro Translation System facilitate enhanced protein folding and solubility for membrane proteins. Additionally, the microgram-scale level of protein expression is thought to prevent an increase in the concentration of nascent protein beyond a threshold that drives aggregation that often plagues bacterial expression. Alternative systems using E. coli extracts typically have a much higher rate of amino acid addition, higher protein yields but have lower membrane concentrations, increasing the chance of protein aggregation by reducing folding efficiency [6,7]. Likewise, in vivo expression methods that yield milligram-scale amounts of membrane proteins often result in the formation of inclusion bodies, which are protein aggregates that are time-intensive and difficult to dissociate into soluble protein.

Membrane proteins are generated by human in vitro translation
The 1-Step Human in vitro Translation System expresses soluble membrane proteins. HA-tagged membrane proteins GypB/E, Bcl2L1 and CXCR4 were expressed using the 1-Step Human Coupled IVT Sytem. Samples (10µL each) were collected from the total reaction mixture (T) and the supernatant (S) and pellet (P) fractions after separation by centrifugation, and the indicated proteins were detected by Western blot analysis using an anti-HA antibody. The figures above the blots are representative of the number of transmembrane domains in each of the proteins. Arrows indicate the respective protein bands.

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Mem-PER Plus Membrane Protein Extraction Kit

Co-express multiple proteins in the same reaction

Co-expression of different proteins can be performed to assess changes in binding affinity due to mutational analysis or small molecule treatment, and the 1-Step Human IVT System can co-express multiple proteins in a single translation reaction. Indeed, as many as five HA-tagged proteins have been expressed in a single reaction using the Human in vitro Translation System.

Multiple proteins can be co-expressed by human in vitro translation
The 1-Step Human in vitro Translation System can co-express multiple proteins. Five HA-tagged proteins were individually translated (as indicated in lanes 1-5) or co-expressed in a single 4-hour reaction at 30°C. The reactions were then separated by 4-12% SDS-PAGE and detected by Western blot using an anti-HA antibody. The proteins were chosen based on their molecular weights to demonstrate the variability in the sizes of the proteins.

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Protein function studies using in vitro-translated protein

References

Cited References:

  1. Jameson J. L. (1998) Principles of molecular medicine. Totowa, N.J.: Humana Press. xix, 1123
  2. Ramachandiran V. et al. (2000) Expression of different coding sequences in cell-free bacterial and eukaryotic systems indicates translational pausing on Escherichia coli ribosomes. FEBS Lett. 482, 185-8.
  3. Endo Y. and Sawasaki T. (2006) Cell-free expression systems for eukaryotic protein production. Curr Opin Biotechnol. 17, 373-80.
  4. Zeenko V. V. et al. (2008) An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by EIF2 phosphorylation. RNA. 14, 593-602.
  5. Maley F. et al. (1989) Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Anal Biochem. 180, 195-204.
  6. Netzer W. J. and Hartl F. U. (1997) Recombination of protein domains facilitated by co-translational folding in eukaryotes. Nature. 388, 343-9.
  7. Madono M. et al. (2010) Wheat germ cell-free protein production system for post-genomic research. N Biotechnol.

 

Product References:

  1. Festa F. et al. (2012) Robust microarray production of freshly expressed proteins in a human milieu. Proteomics Clin Appl. [Epub ahead of print]. Published by Joshua LaBaer's group.
  2. Wang J. et al. (2012) A versatile protein microarray platform enabling antibody profiling against denatured proteins. Proteomics Clin Appl. [Epub ahead of print].
  3. Yadavalli, R., Ledger, C., Sam-Yellowe, TY. (2012). In vitro human cell-free expression system for synthesis of malaria proteins. Parasitol Res. 2012 Jul 11. [Epub ahead of print] DOl I 0.1007 /s00436-0 12-3014-7.
  4. Boohaker R. J. et al. (2011). BAX supports the mitochondrial network, promoting bioenergetics in nonapoptotic cells. Am J Physiol Cell Physiol. 300, C1466-78.
  5. Loughran G. et al. (2011). Ribosomal frameshifting into an overlapping gene in the 2B-encoding region of the cardiovirus genome. Proc Natl Acad Sci U S A. 108, E1111-9.
  6. Stergachis, A. (2011). Rapid empirical discovery of optimal peptides for targeted proteomics. Nature Methods, 8: 1041-1043.
  7. Wang Q. Y. et al. (2011). A translation inhibitor that suppresses dengue virus in vitro and in vivo. Antimicrob Agents Chemother. 55, 4072-80.
  8. Boyne, J. (2010). Kaposi's sarcoma-associated herpesvirus ORF57 protein interacts with PYM to enhance translation of viral intronless mRNAs. EMBO Journal, 29: 1851-1864.
  9. Kasinathan, R., (2010). Schistosoma mansoni express higher levels of multidrug resistance-associated protein 1 (SmMRP1) in juvenile worms and in response to praziquantel. Molecular and Biochemical Parasitology, 173: 25-31.
  10. Khatua, A. (2010). Inhibition of LINE-1 and Alu retrotransposition by exosomes encapsidating APOBEC3G and APOBEC3F. Virology, 400: 68-75.
 

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