Abstract
Based on a previously used plasmid pHC11, a new plasmid pHC11R was constructed. Cutting plasmid pHC11R with proper restriction enzymes, the resulting larger DNA fragment pHC11R’ was co-transformed with a PCR amplified expression cassette of human IFNα2b into yeast. By means of the homologous sequences at both ends of two DNA fragments, a novel expression plasmid pHC11R-IFNα2b was formed via homologous recombination in the yeast. Compared with pHC11-IFNα2b, the expression plasmid pHC11R-IFNα2b was smaller in size and in absence of antibiotic resistant gene. The stability and copy number of pHC11R-IFNα2b were greatly increased and the expression level of heterologous protein was improved. As the derivatives of pHC11R, a series of recombination expression vectors pHRs containing different combination of expression elements were developed. This led to a rapid and powerful method for cloning and expressing of different genes in yeast.
Similar content being viewed by others
References
Brunelli, J. P., Pall, M. L., A series of yeast vectors for expression of cDNAs and other DNA sequences, Yeast, 1993, 9: 1299–1308.
Sikorski, R. S., Hieter, P., A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae, Genetics, 1989, 122: 19–27.
Bonneaud, N., Ozier-Kalogerogoulos, O., Li, G. et al., A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vector, Yeast, 1991, 7: 609–615.
Huo, K. K., Yu, L. L., Chen, X. J., Li, Y. Y., A stable vector for high-level expression and secretion of human interferon alpha A in yeast, Science in China, Ser. B, 1993, 36(5): 557–567.
Zhou, Z. X., Yuan, H. Y., He, W. et al., Expression of the modified HBsAg gene SA-28 directed by a constitutive promoter, Journal of Fudan university (Natural Science), 2000, 39(3): 264–268.
Paques, F., Haber, J. E., Multiple pathways of recombination induces by double-strand breaks in Saccharomyces cerevisiae, Microbiology and Molecular Biology Reviews, 1999, 63(2): 349–404.
Martin, K., Damage-induced recombination in the yeast Saccharomyces cerevisiae, Mutation Research, 2000, 451:91–105.
Alira, S., Tomoko, O., Homologous recombination and the roles of double-strand breaks, TIBS, 1995, 20: 387–391.
Patrick, S., Kelly, M. T., Stephen, V. K., Recombination factor of Saccharomyces cerevisiae, Mutation Research, 2000, 451: 257–275.
Manivasakam, P., Weber, S. C., McElver, J., Schiestl, R. H., Micro-homology mediated PCR targeting in Saccharomyces cerevisiae, Nucleic Acids Res., 1995, 23(14): 2799–2800.
Baudin, A., Lacroute, F., Cullin, C., A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae, Nucleic Acids Res., 1993, 21(14): 3329–3330.
Hua, S. B., Qiu, M., Chan, E., Zhu, L., Luo, Y., Minimum length of sequence homology required for in vivo cloning by homologous recombination in yeast, Plasmid, 1997, 38(2): 91–96.
Hong, M., Sam, K., Peter, J. S. et al., Plasmid construction by homologous recombination in yeast, Gene, 1987, 58: 201–216.
Prado, F., Aguilera, A., New in-vivo cloning methods, methods by homologous recombination in yeast, Curr. Genet., 1994, 20: 180–183.
Jacques, D., DNA insertion system for complex yeast shuttle vectors, Curr. Genet., 1995, 27: 309–311.
Erik, D., Bruno, D., Mireille, D. et al., In vivo cloning by homologous recombination in yeast using a two-plasmid-based system, Yeast, 1995, 11: 629–640.
Kevin, R. O., Kham, T. V., Susan, M., Chris, P., Recombination-mediated PCR-directed plasmid construction in vivo in yeast, Nucleic Acids Res., 1997, 25(2): 451–452.
Falco, S. C., Li, Y. Y., James, R. B., David, B., Genetic properties of chromosomally integrated 2μ plasmid DNA in yeast, Cell, 1982, 29:573–584.
Francesca, S. L., Kevtn, L., Michael, A. R., In vivo site-directed mutagenesis using oligonucleotides, Nature Biotechnology, 2001, 19:773–776.
Chulman, J., Hyuck, K., Sangmee, A. J., In vivo site-directed mutagenesis of yeast plasmids using a three-fragment homologous recombination system, Biotechniques, 2002, 33(2): 288–294.
Wach, A., Brachat, A., Pohlmann, R., Philippsen, P., New heterologus modules for classical or PCR-based gene disruption in Saccharomyces cerevisiea, Yeast, 1994, 10: 1793–1808.
Lorenz, M. C., Muir, R. S., Lim, E. et al., Gene disruption with PCR products in Saccharomyces cerevisiae, Gene, 1995, 158: 113–117.
Bhargava, J., Direct cloning of genomic DNA by recombinogenic targeting method using a yeast-bacterial shuttle vector, pClasper, Genomics, 1999, 62: 285–288.
Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989.
Gietz, R. D., Schiestl, R. H., Williems, A. R. et al., Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure, Yeast, 1995, 11(4): 355–360.
Durke, D., Dawson, D., Stearns, T., Methods in Yeast Genetics. A Cold Spring Harbor Laboratory Course Manual, New York: Cold Spring Harbor Laboratory Press, 2000.
Broach, J. R., Volkert, F. C., Circular DNA plasmids of yeast, in The molecular and cellular biology of the yeast Saccharomyces (eds. Broach, J. R., Pringle, J. R., Jones, E. W.), New York: Cold Spring Harbor Laboratory Press, 1991, Vol. I, 297–331
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Chen, X., Yuan, H., He, W. et al. Construction of a novel kind of expression plasmid by homologous recombination in Saccharomyces cerevisiae . Sci. China Ser. C.-Life Sci. 48, 330–336 (2005). https://doi.org/10.1360/04yc0040
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1360/04yc0040