Express different types of proteins with human in vitro translation

Traditional in vitro translation systems have limitations in the types of proteins that can be generated, because they use either bacterial or insect extracts or lysates from cells that require the addition of ectopic components. The Thermo Scientific Pierce Human in vitro Translation System uses human HeLa or hybridoma lysates that endogenously contain the enzymes required for protein synthesis and proper post-translational modifications. This overview highlights the types of proteins that can be expressed using the Pierce Human in vitro Translation System, which include:

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 Pierce Human in vitro Translation System can be used to produce a broad range of proteins of different sizes.

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

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Human In Vitro Translation Kits and Reagents

pT7CFE1 Expression Vector

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Cell-free In Vitro Recombinant Protein Expression

Getting Started with Human in vitro Translation

Express phosphoproteins with human cell-free extracts

The Thermo Scientific Pierce Human in vitro Translation System uses HeLa or human hybridoma 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 Pierce Human in vitro Translation 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 Pierce Human in vitro Translation 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

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.

The Thermo Scientific Pierce Human in vitro Glycoprotein Expression System uses human hybridoma cell extract instead of rabbit reticulocyte lysate to increase the yield of glycosylated proteins. Hybridoma cells generate antibodies in vivo, all of which are glycosylated to some extent. Because hybridoma cell extracts are rich in ER membranes, they are ideal for driving protein translation that delivers uniform N-glycosylation compared to rabbit reticulocyte lysate supplemented with canine microsomal membranes.

Insect lysates can also be used to generate glycosylated proteins by in vitro translation, although microsomal membranes are not required. While sufficient, the Pierce Human in vitro Glycoprotein Expression System is significantly more efficient at consistently generating glycosylated proteins than insect lysates when comparing optimized amounts of input mRNA and lot-to-lot results. The Human in vitro Glycoprotein Expression System also produces more consistent full-length protein than insect lysates due to the heterogeneity of protein glycoforms generated by insect lysates.

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 Pierce 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 of the Pierce 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.

The Pierce Human in vitro Translation System expresses soluble membrane proteins. HA-tagged membrane proteins GypB/E, Bcl2L1 and CXCR4 were expressed using the Pierce Human in vitro Translation System. Ten-microliter samples 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 Eukaryotic 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 Pierce Human in vitro Translation System can co-express multiple proteins in a single translation reaction. Indeed, we have co-expressed up to 5 HA-tagged proteins were in a single reaction using the Pierce Human in vitro Translation System.

The Pierce 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 that can be co-expressed using the Pierce Human in vitro Translation System.

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Protein function studies using in vitro-translated protein

References