Summary
High-throughput approaches for gene cloning and expression require the development of new, nonstandard tools for use by molecular biologists and biochemists. We have developed and implemented a series of methods that enable the production of expression constructs in 96-well plate format. A screening process is described that facilitates the identification of bacterial clones expressing soluble protein. Application of the solubility screen then provides a plate map that identifies the location of wells containing clones producing soluble proteins. A series of semi-automated methods can then be applied for validation of solubility and production of freezer stocks for the protein production group. This process provides an 80% success rate for the identification of clones producing soluble protein and results in a significant decrease in the level of effort required for the labor-intensive components of validation and preparation of freezer stocks. This process is customized for large-scale structural genomics programs that rely on the production of large amounts of soluble proteins for crystallization trials.
Access provided by Autonomous University of Puebla. Download to read the full chapter text
Chapter PDF
Similar content being viewed by others
Keywords
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
References
Norvell, J.C. and Machalek, A.Z. (2000) Nat. Struct. Biol. 7 Suppl., 931.
Schmid, M.B. (2004) Nat. Rev. Microbiol. 2(9), 739–746.
Zhang, C. and Kim, S.H. (2003) Curr. Opin. Chem. Biol. 7(1), 28–32.
Jung, G.Y. and Stephanopoulos, G. (2004) Science 304, 428–431.
Lee, Y.S. and Mrksich, M. (2002) Trends Biotechnol. 20(12 Suppl.), S14–S18.
Phelan, M.L. and Nock, S. (2003) Proteomics 3(11), 2123–2134.
Lehner, B. and Fraser, A.G. (2004) Genome Biol. 5(9), R63.
Terradot, L. Durnell, N., Li, M., Li, J., Ory, J., Labigne, A., Legrain, P., Colland, F. and Waksman, G. (2004) Mol. Cell. Proteomics 3(8), 809–819.
Chambers, S.P. (2002) Drug Discov. Today 7(14), 759–765.
Endo, Y. and Sawasaki, T. (2003) Biotechnol. Adv. 21(8), 695–713.
Sarafanov, A. and Saenko, E. (2004) Anal. Biochem. 328(1), 98–100.
Lesley, S.A. (2001) Protein Expr. Purif. 22(2), 159–164.
Dieckman, L., Gu, M., Stols, L., Donnelly, M.I. and Collart, F.R. (2002) Protein Expr. Purif. 25(1), 1–7.
Savchenko, A., Yee, A., Khachatryan, A., Skarina, T., Evdokimova, E., Pavlova, M., Semesi, A., Northey, J., Beasley, S., Lan, N., Das, R., Gerstein, M., Arrowmith, C.H. and Edwards, A.M. (2003) Proteins 50(3), 392–399.
Yee, A., Pardee, K., Christendat, D., Savchenko, A., Edwards, A.M. and Arrowsmith, C.H.. (2003) Acc. Chem. Res. 36(3), 183–189.
Baneyx, F. (1999) Curr. Opin. Biotechnol. 10(5), 411–421.
Baneyx, F. and Palumbo, J.L. (2003) Methods Mol. Biol. 205, 171–197.
Chance, M.R., Fiser, A., Sali, A., Pieper, U., Eswar, N., Xu, G., Fajardo, J.E., Radhakannan, T. and Marinkovic, N. (2004) Genome Res. 14(10B), 2145–2154.
Folkers, G.E., van Buuren, B.N. and Kaptein, R. (2004) J. Struct. Funct. Genomics 5(1–2), 119–131.
Yokoyama, S. (2003) Curr. Opin. Chem. Biol. 7(1), 39–43.
Baneyx, F. (1999) Curr. Opin. Biotechnol. 10(5), 411–421.
Chambers, S.P., Austen, D.A., Fulghum, J.R. and Kim, W.M. (2004) Protein Expr. Purif. 36(1), 40–47.
Laage, R. and Langosch, D. (2001) Traffic 2(2), 99–104.
Uchimura, Y., Nakamura, M., Sugasawa, K., Nakao, M. and Saitoh, H. (2004) Anal. Biochem. 331(1), 204–206.
DiDonato, M., Deacon, A.M., Klock, H.E., McMullan, D. and Lesley, S.A. (2004) J. Struct. Funct. Genomics 5(1–2), 133–146.
Janda, I., Devedjiev, Y., Cooper, D., Chruszcz, M., Derewenda, U., Gabrys, A., Minor, W., Joachimiak, A. and Derewenda, Z.S. (2004) Acta Crystallogr. D Biol. Crystallogr. 60 (Pt 6), 1101–1107.
Lundstrom, K. (2004) Comb. Chem. High Throughput. Screen. 7(5), 431–439.
Haun, R.S., Serventi, I.M. and Moss, J. (1992) Biotechniques 13(4), 515–518.
Aslanidis, C. and de Jong, P.J. (1990) Nucl. Acids Res. 18(20), 6069–6074.
Yoon, J.R., Laible, P.D., Gu, M., Scott, H.N. and Collart, F.R. (2002) Biotechniques 33(6), 1328–1333.
di Guan, C., Li, P., Riggs, P.D. and Inouye, H. (1988) Gene 67(1), 21–30.
Routzahn, K.M. and Waugh, D.S. (2002) J. Struct. Funct. Genomics 2(2), 83–92.
Stols, L., Gu, M., Dieckman, L., Raffen, R., Collart, F.R. and Donnelly, M.I. (2002) Protein Expr. Purif. 25(1), 8–15.
Woestenenk, E.A., Hammarstrom, M., van den Berg, S., Hard, T. and Berglund, H. (2004) J. Struct. Funct. Genomics 5(3), 217–229.
Moy, S., Dieckman, L., Schiffer, M., Maltsev, N., Yu, G.X. and Collart, F.R. (2004) J. Struct. Funct. Genomics 5(1–2), 103–109.
Nguyen, H., Martinez, B., Oganesyan, N. and Kim, R. (2004) J. Struct. Funct. Genomics 5(1–2), 23–27.
Finley, J.B., Qiu, S.H., Luan, C.H. and Luo, M. (2004) Protein Expr. Purif. 34(1), 49–55.
Coleman, M.A., Lao, V.H., Segelke, B.W. and Beernink, P.T. (2004) J. Proteome Res. 3(5), 1024–1032.
Shih, Y.P., Kung, W.M., Chen, J.C., Yeh, C.H., Wang, A.H. and Wang, T.F. (2002) Protein Sci. 11(7), 1714–1719.
Hammarstrom, M., Hellgren, N., van Den Berg, S., Berglund, H. and Hard, T. (2002) Protein Sci. 11(2), 313–321.
Lesley, S.A., Graziano, J., Cho, C.Y., Knuth, M.W. and Klock, H.E. (2002) Protein Eng. 15(2), 153–160.
Waldo, G.S. (2003) Curr. Opin. Chem. Biol. 7(1), 33–38.
Millard, C.S., Stols, L., Quartey, P., Kim, Y., Dementieva, I. and Donnelly, M.I. (2003) Protein Expr. Purif. 29(2), 311–320.
Prinz, B., Schultchen, J., Rydzewski, R., Holz, C., Boettner, M., Stahl, U. and Lang, C. (2004) J. Struct. Funct. Genomics 5(1–2), 29–44.
Stols, L., Millard, C.S., Dementieva, I. and Donnelly, M.I. (2004) J. Struct. Funct. Genomics 5(1–2), 95–102.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Springer Science+Business Media, Inc.
About this chapter
Cite this chapter
Dieckman, L.J., Hanly, W.C., Collart, F.R. (2006). Strategies for High-Throughput Gene Cloning and Expression. In: Setlow, J.K. (eds) Genetic Engineering. Genetic Engineering: Principles and Methods, vol 27. Springer, Boston, MA. https://doi.org/10.1007/0-387-25856-6_10
Download citation
DOI: https://doi.org/10.1007/0-387-25856-6_10
Publisher Name: Springer, Boston, MA
Print ISBN: 978-0-387-25855-3
Online ISBN: 978-0-387-25856-0
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)