Application of In Vitro Protein Expression to Human Prote
- Molecular & Structural Biology Division, Lawrence Livermore National Laboratory, 7000 East Ave., Livermore, CA 94550,, USA
- Roche Diagnostics Corp., 2929 7th St., Ste. 100, Berkeley, CA 94710, USA
INTRODUCTION. A critical step in many proteomics projects is the identification of genes that express proteins in sufficient quantities for subsequent studies. LLNL houses a collection of >200,000 human cDNAs, known as the IMAGE Consortium (http://image.llnl.gov), including some full-length enriched cDNA libraries. We have developed an integrated system that exploits coupled in vitro transcription/translation (IVT) to identify human IMAGE cDNAs that express proteins of the predicted mass. Moreover, this expression screening can be conducted without plasmid DNA preparation or subcloning. IVT-produced proteins are used directly in functional assays or the cDNAs are transported to a bacterial expression system for large-scale protein production. METHODS. IVT reactions are performed using the RTS-500 Rapid Translation System (Roche Molecular Biochemicals) in conjunction with a biotin-lysine-tRNA conjugate, FluoroTectTM GreenLys (Promega). IVT products are visualized on a UV transilluminator or quantitated on a Packard fluorescence plate reader (λex = 485 nm; λem = 510 nm). For micro- and far-western blots, 100 nl of IVT-produced protein are spotted on a glass slide. Detection is accomplished using adapted protocols and a rhodamine-conjugated secondary antibody. Crystallization experiments are conducted by the LLNL TB Crystallization Center (http://www-structure.llnl.gov/TB) using CrysTool software (2) interfaced with Packard MultiProbeII liquid handling robots. RESULTS. We have performed preliminary studies to optimize steps in small-scale IVT screening. We have compared Escherichia. coli extracts from several different vendors. Using the extract of choice, we have obtained similar protein yields using plasmid and PCR template DNA. We have scaled reactions down to 12 µl volumes, which results in significant cost savings in both RTS extracts and FluoroTectTM GreenLys. Finally, we have optimized the ratio of labeled to unlabeled lysine in the reactions. The yields from in vitro and in vivo protein expression correlate well, which validates the use of IVT for expression screening. For large-scale expression, the cDNAs are subcloned into a modified version of pETBlue-2 (Novagen) for high-level expression of C-terminally His-tagged proteins.
- Research Organization:
- Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division
- Grant/Contract Number:
- W-7405-Eng-48; AC52-07NA27344
- OSTI ID:
- 1198078
- Alternate ID(s):
- OSTI ID: 1629121
- Journal Information:
- The Scientific World Journal (Online), Journal Name: The Scientific World Journal (Online) Vol. 2; ISSN 1537-744X
- Publisher:
- Hindawi Publishing CorporationCopyright Statement
- Country of Publication:
- United Kingdom
- Language:
- English
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