Ambergen Technology

In combination with cell-free (in vitro) protein expression, tRNA mediated protein engineering (TRAMPE) can be used to co-translationally incorporate non-native amino acids into the nascent proteins by including special misaminoacylated tRNAs in the reaction [1-7].

Ambergen has developed a novel class of proprietary misaminoacylated tRNAs, in particular, capable of incorporating fluorescent and/or photocleavable biotin (PC-Biotin) labeled amino acids, both at the N-terminus or randomly throughout the peptide chain, using prokaryotic or eukaryotic expression systems (including mammalian) [8-11] (Figure 1). Ambergen licenses one such tRNA, FluoroTect™ GreenLys, to Promega Corporation for research reagent sales. Moreover, as reported by Iborra et al. in Science, this tRNA has also proven useful in permeabilized mammalian cells for basic research applications [12].

Applications of TRAMPE

Cell-free protein production in general is extremely rapid, e.g. hours, as well as simple, e.g. no cellular transfection or cell culture. Cell-free expression is also compatible with PCR templates, i.e. gene cloning is compatible but not required, which can benefit certain screening applications. Coupled with the ability to incorporate fluorescence detection labels and/or photocleavable affinity tags make cell-free expression a powerful tool for a host of applications, such as pull-down assays for measuring protein-protein interaction (Figure 2) or both a gel-based and a novel high throughput ELISA-based non-isotopic protein truncation test (PTT) for mutation detection in molecular diagnostics of inherited diseases. Further details on this application to molecular diagnostics can be found in Ambergen's 2003 Nature Biotechnology publication [9].

Finally, these TRAMPE technologies have utility in the area proteomics, particularly the production of cell-free expressed protein libraries, arrays and bead-displays. Such areas are under development at Ambergen for use in biomarker discovery and detection, for applications in diagnostics, prognostics and theranostics.

TRAMPE and Protein Purification

Figure 3 illustrates the power of TRAMPE for protein labeling and purification. A GST test protein was cell-free expressed and TRAMPE labeled with both fluorescence and PC-Biotin, as depicted in Figure 1. The protein was then isolated on (strept)avidin beads via its directly incorporated PC-Biotin, and photo-released in pure form, an approach termed PC-SNAG. A parallel and more conventional isolation of the same protein was achieved using immobilized metal affinity chromatography (IMAC) via the polyhisitidine tag also present in the GST. Fluorescence SDS-PAGE shows the yields of the 2 methods are comparable. However, high sensitivity silver staining of the same gel, to detect total protein, reveals several contaminants in the polyhistidine method, of equal or greater intensity to the target protein. In contrast, the TRAMPE based PC-SNAG method shows far superior purity.

References

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2. Forster, A.C., ed. Engineering Translation. Methods. Vol. 36 (3). 2005. 225-320.
3. Krieg, U.C., A.E. Johnson, and P. Walter, Protein translocation across the endoplasmic reticulum membrane: identification by photocross-linking of a 39-kD integral membrane glycoprotein as part of a putative translocation tunnel. J Cell Biol, 1989. 109(5): p. 2033-43.
4. Sonar, S., C.P. Lee, M. Coleman, N. Patel, X. Liu, T. Marti, H.G. Khorana, U.L. Rajbhandary, and K. Rothschild, Site-directed isotope labelling and FTIR spectroscopy of bacteriorhodopsin. Nature Structural Biology, 1994. 1(8): p. 512-517.
5. Noren, C.J., S.J. Anthony-Cahill, M.C. Griffith, and P.G. Schultz, A general method for site-specific incorporation of unnatural amino acids into proteins. Science, 1989. 244(4901): p. 182-8.
6. Rothschild, K.J. and S. Gite, tRNA-mediated protein engineering. Curr Opin Biotechnol, 1999. 10(1): p. 64-70.
7. Rothschild, K.J., S. Gite, S. Mamaev, and J. Olejnik, Building Photonic Proteins, in CRC Handbook of Organic Photochemistry and Photobiology, F. Lenci, Editor. 2003. p. Chapter 133, pages 1-21.
8. Gite, S., S. Mamaev, J. Olejnik, and K. Rothschild, Ultrasensitive fluorescence-based detection of nascent proteins in gels. Anal Biochem, 2000. 279(2): p. 218-25.
9. Gite, S., M. Lim, R. Carlson, J. Olejnik, B. Zehnbauer, and K. Rothschild, A high-throughput nonisotopic protein truncation test. Nat Biotechnol, 2003. 21(2): p. 194-7.
10. Mamaev, S., J. Olejnik, E.K. Olejnik, and K.J. Rothschild, Cell-free N-terminal protein labeling using initiator suppressor tRNA. Anal Biochem, 2004. 326(1): p. 25-32.
11. Olejnik, J., S. Gite, S. Mamaev, and K.J. Rothschild, N-terminal labeling of proteins using initiator tRNA. Methods, 2005. 36(3): p. 252-60.
12. Iborra, F.J., D.A. Jackson, and P.R. Cook, Coupled transcription and translation within nuclei of mammalian cells. Science, 2001. 293(5532): p. 1139-42.