title: Analysis of prepro-alpha-lytic protease expression in Escherichia coli reveals that the pro region is required for activity.
abstract: The alpha-lytic protease of Lysobacter enzymogenes was successfully expressed in Escherichia coli by fusing the promoter and signal sequence of the E. coli phoA gene to the proenzyme portion of the alpha-lytic protease gene. Following induction, active enzyme was found both within cells and in the extracellular medium, where it slowly accumulated to high levels. Use of a similar gene fusion to express the protease domain alone produced inactive enzyme, indicating that the large amino-terminal pro region is necessary for activity. The implications for protein folding are discussed. Furthermore, inactivation of the protease by mutation of the catalytic serine residue resulted in the production of a higher-molecular-weight form of the alpha-lytic protease, suggesting that the enzyme is self-processing in E. coli.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
authors.forename: J L
authors.initials: JL
authors.lastname: Silen
authors.forename: D
authors.initials: D
authors.lastname: Frank
authors.forename: A
authors.initials: A
authors.lastname: Fujishige
authors.forename: R
authors.initials: R
authors.lastname: Bone
authors.forename: D A
authors.initials: DA
authors.lastname: Agard
mesh_terms: D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006090:Gram-Negative Bacteria / Q000201:enzymology* / Q000235:genetics; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D004792:Enzyme Precursors; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C058755:prepro-alpha-lytic protease; C021826:myxobacter alpha-lytic proteinase