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The Chemical Synthesis of a Gene Coding for Bovine Pancreatic DNase I and Its Cloning and Expression in Escherichia Coli

The Chemical Synthesis of a Gene Coding for Bovine Pancreatic DNase I and Its Cloning and Expression in Escherichia Coli

A F Worrall, B A Connolly

J Biol Chem. 1990 Dec 15;265(35):21889-95.

PMID: 2254338

Abstract:

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP9003989-A	Deoxyribonuclease I from bovine pancreas		9003-98-9	Price

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