title: Efficient E. coli expression systems for the production of recombinant β-mannanases and other bacterial extracellular enzymes.
abstract: Two Escherichia coli expression systems based on T7 RNA polymerase promoter (pET system) and tac promoter (pFLAG system) have been used for the production and secretion of recombinant β-mannanases from Bacillus sp. Both E. coli OmpA signal peptide and native Bacillus signal peptide could be used efficiently for the secretion of recombinant enzymes into periplasmic space and culture media. The genes could be induced for over-expression with 0.1-1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) when the OD 600 of the culture broth reached 0.6-1.5. The recombinant enzymes could be harvested from whole cell lysate, perimplasmic extract, or culture broth after induction for 4-20 hours. Since the enzyme is C-terminally tagged with hexahistidine, the recombinant enzymes could be conveniently purified to apparent homogeneity by one-step immobilized-metal affinity chromatography (IMAC) using Ni-NTA resins. The characteristics of purified recombinant β-mannanases from B. licheniformis and B. subtilis, which share 78% amino acid identity, are slightly different. These systems should be applicable for the production of various recombinant bacterial extracellular enzymes.
authors.affiliation: School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand. montarop@sut.ac.th
authors.forename: Montarop
authors.initials: M
authors.lastname: Yamabhai
authors.forename: Bancha
authors.initials: B
authors.lastname: Buranabanyat
authors.forename: Nanthnit
authors.initials: N
authors.lastname: Jaruseranee
authors.forename: Chomphunuch
authors.initials: C
authors.lastname: Songsiriritthigul
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D008957:Models, Genetic; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D044902:beta-Mannosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D011994:Recombinant Proteins; C078167:OMPA outer membrane proteins; D044902:beta-Mannosidase