Nucleic Acids Research, 1992, Vol. 20, No. 12 3127-3133
© 1992
MOLECULAR BIOLOGY |
Generating compatible translation initiation regions for heterologous gene expression in Escherichia coli by exhaustive periShine-Dalgarno mutagenesis. Human glutathione reductase cDNA as a model
Institut für Biochemie II, Universität Heidelberg Heidelberg, Germany 1Institut für Zell- und Tumorbiobgie, Deutsches Krebsforschungszentrum, Heidelberg, Germany
*To whom correspondence should be addressed at: Institut für Biochemie II, Germany Universiut Heidelberg, Im Neuenheimer Feld 328, D-6900 Heidelberg, Germany
Received February 26, 1992. Revised May 19, 1992. Accepted May 19, 1992.
Adaptation of eucaryotic cDNA to heterologous expression was studied by mutating the translation initiation (TI) region upstream (mTI) and downstream (MTI) of the start codon. in the mTI subregion the 8 bases flanking the invariant Shine-Daigarno motif GG-AG were mutagenized exhaustively, whiie the MTI subregion was subjected to random silent mutations at the wobbie positions. The quaiity of a given TI sequence was judged on the basis of expressed enzyme activity. Low-yield and high-yield mutants of both TI subregions were selected and recombined systematically. The analysis of these double cartridges gave the following results: 1. As a rule, an unfavourable MTI subregion can be compensated for by mutations in the mTI subregion and vice versa. 2. The compatibility between mT1 and MTI subregion is explainable at least in part by a low interaction tendency; a
G°'-value of -10.7 kcal/mol appears to be a physical threshold for heterologous cDNA expression. 3. On the basis of periShine-Dalgamo mutations, the expression yield for different cDNA sequences could be increased by 1 to 2 orders of magnitude. One of these sequences encoded (1-15)human glutathione reductase, a mutant lacking the flexible N-terminal extension of the protein. In conclusion, to study and overcome TI region-based expression problems it is worthwhile to start out with a versatile vector containing exhaustive mutations in the periShine-Dalgarno sequences; as a rule the coding MTI subregion can be kept unchanged.
+Present address: Dr.Karl Thomae GmbH. Biotechnicai Production, D-7950 Biberach, Germany
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