The chemical synthesis of a gene coding for bovine pancreatic DNase I and its cloning and expression in Escherichia coli.
A gene coding for bovine pancreatic DNase I has been constructed from synthetic oligonucleotides. This gene has been cloned into a plasmid vector pDOC55 designed to allow very tight control of expression of potentially lethal proteins. Induction of protein synthesis from the gene yielded a peptide of molecular weight of approximately 31,000, consistent with DNase I. The yield of this protein from the pDOC55 construct (pAW5) was approximately 150 micrograms/liter of cell culture. Attempts to clone the gene into a less tightly controlled expression vector based on the tac-promoter (pKK223-3) were unsuccessful, presumably due to the expected lethality of the product. Mutagenesis of the gene to replace the active site histidine (His-134) in the protein with glutamine yielded a gene readily clonable into both expression systems. Yields of the mutagenized protein were approximately 6 micrograms/liter from a pDOC55 system and 20 mg/liter from a pKK223-3 system. The activity of the proteins were assayed using the Kunitz procedure and their cleavage selectivities by digestion of the Escherichia coli tyr T promoter. The recombinant native enzyme had both the same specific activity and DNA cleavage selectivity as the protein isolated from bovine pancreas using these two assays. The H134Q mutant had a specific activity of about 0.001% of the native protein but had an unaltered DNA cleavage selectivity.
Worrall
A F
AF
Department of Biochemistry, University of Southampton, United Kingdom.
Connolly
B A
BA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005796:Genes; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity
D004269:DNA, Bacterial; D003850:Deoxyribonuclease I