By Goeddel D.V., Emr S.D., Gold L. (eds.)

The tools provided during this quantity will permit the reader to layout potent innovations for the expression of cloned genes and cDNAs and may turn out beneficial in fixing the vast majority of expression difficulties one is probably going to come across.

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3] DESIGN AND CONSTRUCTION OF PLASMID VECTORS 35 The genes contained in the plasmid will also determine plasmid stability and the overall product yield as well. Cloning and expression vectors usually contain at least one gene for selection which, as discussed earlier, has to exert a beneficial effect to avoid segregation. Manipulation of Plasmid DNA Because plasmids are one of the central tools in molecular cloning, a number of efficient methods for their purification, storage, mutagenesis and transformation of hosts have been developed.

Rev. Biochem. 43, 721 (1974). 3 W. R. McClure, Annu. Rev. Biochem. 54, 171 (1985). 4 W. Gilbert, in "RNA Polymerase" (R. Losick and M. ), p. 193. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1976. 50. Raibaud and M. Schwartz, Annu. Rev. Genet. 18, 173 (1984). 6 M. Rosenberg and D. Court, Annu. Rev. Genet. 13, 319 (1979). 7 U. Siebenlist, R. B. Simpson, and W. Gilbert, Cell 20, 269 (1980). s T. D. Yager and P. H. yon Hippel, in "Escherichia coil and Salmonella typhimurium'" (F. C.

Therefore, this region may be manipulated to maximize translation initiation. In contrast to the limited number of inducible promoters that are used in expression plasmid vectors, a large variety of regions for translation initiation are in use. Although codon usage has often been assigned a crucial role in determining the efficiency of protein synthesis, there is now growing evidence that this is not necessarily the case. 71 The issue of protein degradation represents a particularly difficult problem that may be encountered when overproducing a protein in E.

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