Moving Out of the Cell: Advantages of Cell-Free Protein Expression

Cell-free protein expression is a simplified and accelerated avenue for the transcription and/or translation of a specific protein in a quasi cell environment. An alternative to slower, more cumbersome cell-based methods, cell-free protein expression methods are simple and fast and can overcome toxicity and solubility issues sometimes experienced in the traditional E. coli expression systems.

Cell-free protein expression offers significant time savings over cell-based expression methods.
Cell-free protein expression offers significant time savings over cell-based expression methods.

Continue reading “Moving Out of the Cell: Advantages of Cell-Free Protein Expression”

Cell-free Expression: A System for Every Need

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Cell-free protein expression is a simplified and accelerated avenue for the transcription and/or translation of a specific protein in a quasi cell environment. An alternative to slower, more cumbersome cell-based methods, cell-free protein expression methods are simple and fast and can overcome toxicity and solubility issues sometimes experienced in traditional E. coli expression systems.

In his webinar, “In vitro, cell-free protein expression–How it helps speed up your research”, Gary Kobs offered an overview of the different cell-free expressions systems offered by Promega and highlighted what needs the different systems best address. He discussed different applications of cell-free expressed proteins and highlighted the combined uses of the HaloTag® Technology with cell-free protein expression. Continue reading “Cell-free Expression: A System for Every Need”

High-Yield Cell-Free Protein Expression: Prokaryotic Based

S30 E coli high yield extract schematicMany applications require amounts of protein that cannot be obtained using a eukaryotic cell-free expression system. As an alternative, a prokaryotic system can be used when this need arises. The E. coli S30 T7 High-Yield Protein Expression System is designed to express up to 500μg/ml of protein in 1 hour from plasmid vectors containing a T7 promoter and a ribosome binding site. The protein expression system provides an extract that contains T7 RNA polymerase for transcription and is deficient in OmpT endoproteinase and lon protease activity. All other necessary components in the system are optimized for protein expression. This results in greater stability and enhanced expression of target proteins.The following references highlight the use of this system for a variety of unique applications:

Loh, E. et al. (2011) An unstructured 5′-coding region of the prfA mRNA is required for efficient translation. Nuc. Acids. Res. (online) Examines the effect of upstream codon sequence/length on the correct ribosome binding and translation initiation of the pfrA protein.

Mitsuhashi, H. et al. (2010) Specific phosphorylation of Ser458 of A-type lamins in LMNA-associated myopathy patients. J. Cell. Sci. 123, 3893–900 By creating a series of mutations in the protein lamin A, Akt1 phosphorylation sites were determined.

Halvorsen, E. et al. (2011) Txe, an endoribonuclease of the enterococcal Axe-Txe toxin-antitoxin system, cleaves mRNA and inhibits protein synthesis. Microbiology 157, 387–97. S30 High Yield System was used to characterize the inhibitory effect of Txe toxin on protein expression.

Mo, P. et al. (2010) MDM2 mediates ubiquitination and degradation of activating transcription factor 3. J. Biol. Chem. 285, 26908–15. By using in vitro pull down experiments the researchers characterized the binding of AFT3 to MDM2 leading to the proteolysis of AFT3 system by ubiquitination.

Optimized Protein Expression: Flexi Rabbit Reticulocyte Lysate

A protein chain being produced from a ribosome.

mRNAs commonly exhibit differing salt requirements for optimal translation. Small variations in salt concentration can lead to dramatic differences in translation efficiency. The Flexi® Rabbit Reticulocyte Lysate System allows translation reactions to be optimized for a wide range of parameters, including
Mg2+ and K+ concentrations and the choice of adding DTT. To help optimize Mg2+ for a specific message, the endogenous Mg2+ concentration of each lysate batch is stated in the product information included with this product.

The following references utilize the features of Flexi Rabbit Reticulocyte Lysate System to investigate certain parameters of translation:

Vallejos, M. et al. (2010)The 5′-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation. Nucl Acids Res. 38, 618–32. Identification of internal ribosomal ribosomal entry site in the 5’ untranslated region of the mouse mammary tumor virus mRNA.

Spriggs, K. et al. (2009) The human insulin receptor mRNA contains a functional internal ribosome entry segment. Nucl. Acids. Res. 17, 5881–93. Identification of a functional internal ribosome entry site in the human insulin receptor mRNA.

Powell, M. et al. (2008) Characterization of the termination-reinitiation strategy employed in the expression of influenza B virus BM2 protein. RNA 14, 2394–06. Analysis of the mRNA signals involved in the expression of influenza B virus BM2 protein.

Sato, V. et al. (2007) Measles virus N protein inhibits host translation by binding to eIF3-p40. J. Vir. 81, 11569–76. Charaterized the effect of the measles virus N protein binding to the translation initiation factor eIF3-p40 on the expression of various proteins in rabbit reticulocyte lysate.

Hirao, K. et al. (2006) EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming. J. Biol. Chem. 281, 9650–58. The EDEM3 protein was expressed in the presence of canine microsomal membranes to establish that co-translational translocation occurs into the endoplasmic reticulum.

Shenvi, C. et al. (2005) Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations–implications for the study of ribosome dynamics. RNA 11, 1898–08. Characterization of ribosome dynamics under different ionic conditions.

Nair, A. et al. (2005) Regulation of luteinizing hormone receptor expression: evidence of translational suppression in vitro by a hormonally regulated mRNA-binding protein and its endogenous association with luteinizing hormone receptor mRNA in the ovary. J. Biol. Chem. 280, 42809–16. Examined the affect of luteinizing hormone receptor mRNA binding protein on transltional suppression of luteinizing hormone receptor RNA.

Cell-Free Expression Applications: Characterization of Viral-Mediated Diseases

INFECTION IS BY TRANSCRIPTION OF THE VIRAL DNA INTO THE GENETIC MATERIAL OF THE HOST CELL, SO ANTIBODY TEST ING CAN DIAGNOSE AIDS. MORE SIGNIFICANTLY, IF THE PERCENTAGE OF LYMPHOCYTES THAT ARE CD4+ IS ABOVE 500 PER CUBIC MM THERE IS BETTER PROGNOSIS FOR TREATMENT. HERE THE VIRUS IS BUDDING FROM THE T CELL AS PART OF ITS PROLIFERATION AFTER INFECTING THE T CELL. AIDS VIRUS DIGITIZED TEM COLOR ENHANCED

The characterization of viral mediated diseases is critical to promote the overall welfare of humans or animals. Initial research focused on the interpretation the genomic content (i.e., DNA or RNA based) of the selected virus. The next step is to better understand the proteins that are encoded by this content and their interaction with the host proteome.
The following citations illustrate the use of cell-free protein expression to facilitate this research. Continue reading “Cell-Free Expression Applications: Characterization of Viral-Mediated Diseases”

Cell Free Expression Application: Production of Soluble Protein for Structural Analysis

The TNT® SP6 High-Yield Protein Expression System uses a high-yield wheat germ extract supplemented with SP6 RNA polymerase and other components. Coupling transcriptional and translational activities eliminates the inconvenience of separate in vitro transcription and purification steps for the mRNA, while maintaining the high levels of protein expression. All that is required is the addition of DNA templates containing the SP6 promoter and the protein coding region for the protein of interest. Furthermore no specialized equipment is required for protein screening and production. The system enables the expression of approximately 100µg/ml of protein in batch reaction and 200–440µg/ml in dialysis reaction in 10–20 hours .

In a recent publication (Zhao, L. et.al. (2010) J. Struct. Genomics 11, 201–9), the Northeast Structural Genomics Consortium (www.nesg.org) in their quest to express 5,000 eukaryotic proteins, report that even with different cloning strategies they could only produce 26% of the proteins in a soluble form. To improve the efficiency of expressing soluble protein, they investigated the use of wheat germ cell free system as a alternative to E.coli.

In this publication 59 human constructs were expressed in both E.coli and the wheat germ cell free system. Only 30% of human proteins could be produced in a soluble form using E.coli -based expression. Some 70% could be produced using the TNT® SP6 High Yield Wheat Germ system.
To further demonstrate the utility of expressing proteins that were suitable for structural studies from a wheat germ-based system, two of the proteins were isotope enriched and analyzed successfully by 2D NMR.

Screening for Protein Activity Using Cell-Free Expression

The analysis of functional protein typically requires lengthy laborious cell based protein expression that can be complicated by the lack of stability or solubility of the purified protein. Cell free protein expression eliminates the requirement for cell culture thus providing quick access to the protein of interest (1).

The HaloTag® Technology provides efficient, covalent and oriented protein immobilization of the fusion protein to solid surfaces (2).

A recent publication demonstrated the feasibility of using cell free expression and the HaloTag technology to express and capture a fusion protein for the rapid screening of protein kinase activity (3). The catalytic subunit of human cAMP dependent protein kinase was expressed in a variety of cell free expression formats as a HaloTag fusion protein. The immobilized cPKA fusion protein was assayed directly on magnetic beads in the active form and was shown to be inhibited by known PKA inhibitory compounds.

Therefore this unique combination of protein expression and capture technologies can greatly facilitate the process of activity screening and characterization of potential inhibitors

References
ResearchBlogging.org

  1. Zhao, K.Q. et al. (2007) Functional protein expression from a DNA based wheat germ cell-free system. J. Struc. Funct. Genomics. 8, 199-208.
  2. Los, G.V. and Wood, K. (2007) The HaloTag: A novel technology for cell imaging and protein analysis. Meth. Mol. Biol. 356, 195-208
  3. Leippe DM, Zhao KQ, Hsiao K, & Slater MR (2010). Cell-free expression of protein kinase a for rapid activity assays. Analytical chemistry insights, 5, 25-36 PMID: 20520741

6X His Protein Pulldowns: An Alternative to GST

ResearchBlogging.orgPull-down assays probe interactions between a protein of interest that is expressed as fusion protein (e.g.,
(e.g., bait) and the potential interacting partners (prey).

In a pull-down assay one protein partner is expressed as a fusion protein (e.g., bait protein) in E. coli and then immobilized using an affinity ligand specific for the fusion tag. The immobilized
bait protein can then be incubated with the prey protein. The source of the prey protein depends on whether the experiment is designed to confirm an interaction or to identify new interactions. After a series of wash steps, the entire complex can be eluted from the affinity support using SDS-PAGE loading buffer or by competitive analyte elution, then evaluated by SDS-PAGE.

Successful interactions can be detected by Western blotting with specific antibodies to both the prey and bait proteins, or measurement of radioactivity from a [35S] prey protein. bait) and potential interacting partners (prey).

The most commonly used method to generate a bait protein is expression as a fusion protein contain a GST (glutathione-S transferase) tag in E. coli. This is followed by immobilization on particles that contain reduced glutathione, which binds to the GST tag of the fusion protein. The primary advantage of a GST tag is that it can increase the solubility of insoluble or semi-soluble proteins expressed in E. coli.

Among fusion tags, His-tag is the most widely used and has several advantages including: 1) It’s small in size, which renders it less immunogenically active, and often it does not need to be removed from the purified protein for downstream applications; 2) There are a large number of commercial vectors available for expressing His-tagged proteins; 3) The tag may be placed at either the N or C terminus; 4) The interaction of the His-tag does not depend on the tag structure, making it possible to purify otherwise insoluble proteins using denaturing conditions. Continue reading “6X His Protein Pulldowns: An Alternative to GST”

Optimized Wheat Germ Extract for High-Yield Protein Expression of Functional, Soluble Protein

proteinexpressionCell-free protein synthesis has emerged as powerful alternative to cell based protein expression for functional and structural proteomics. The TNT® SP6 High-Yield Protein Expression System uses a high-yield wheat germ extract supplemented with SP6 RNA polymerase and other components. Coupling transcriptionaland translational activities eliminates the inconvenience of separate in vitro transcription and purification steps for the mRNA, while maintaining the high levels of protein expression (1). Continue reading “Optimized Wheat Germ Extract for High-Yield Protein Expression of Functional, Soluble Protein”