title: Secretory expression in Escherichia coli and single-step purification of a heat-stable alkaline protease.
abstract: Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli cytoplasm into the culture medium. The gene for a highly thermostable alkaline protease was cloned from Bacillus stearothermophilus F1 by the polymerase chain reaction. The recombinant F1 protease was efficiently excreted into the culture medium using E. coli XL1-Blue harboring two vectors: pTrcHis bearing the protease gene and pJL3 containing the BRPs. Both vectors contain the E. coli lac promoter-operator system. In the presence of 40 microM IPTG, the recombinant F1 protease and the BRP were expressed and mature F1 protease was released into the culture medium. This opens the way for the large-scale production of this protease in E. coli. The recombinant enzyme was purified through a one-step heat treatment at 70 degrees C for 3h and this method purified the protease to near homogeneity. The purified enzyme showed a pH optimum of 9.0, temperature optimum of 80 degrees C, and was stable at 70 degrees C for 24h in the pH range from 8.0 to 10.0. The enzyme exhibited a high degree of thermostability with a half-life of 4 h at 85 degrees C, 25 min at 90 degrees C, and was inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF).
authors.affiliation: Department of Biochemistry, Hongkong University of Science and Technology, 43400 UPM Serdang, Selangor, Malaysia.
authors.forename: Zhibiao
authors.initials: Z
authors.lastname: Fu
authors.forename: Suhali Bt Ab
authors.initials: SB
authors.lastname: Hamid
authors.forename: Che Nyonya A
authors.initials: CN
authors.lastname: Razak
authors.forename: Mahiran
authors.initials: M
authors.lastname: Basri
authors.forename: Abu Bakar
authors.initials: AB
authors.lastname: Salleh
authors.forename: Raja Noor Zaliha Abd
authors.initials: RN
authors.lastname: Rahman
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C032923:microbial serine proteinases