title: Temperature-regulated expression of the tac/lacl system for overproduction of a fungal xylanase in Escherichia coli.
abstract: Temperature-regulated expression of recombinant proteins in the tac promoter (Ptac) system was investigated. Expression levels of fungal xylanase and cellulase from N. patriciarum in E. coli strains containing the natural lacI gene under the control of the Ptac markedly increased with increasing cultivation temperature in the absence of a chemical inducer. The specific activities (units per milligram protein of crude enzyme) of the fungal xylanase and cellulase produced from recombinant E. coli strain pop2136 grown at 42 degrees C were about 4.5 times higher than those of the cells grown at 23 degrees C and were even slightly higher when compared with cells grown in the presence of the inducer isopropyl beta-D-thiogalactopyranoside. The xylanase expression level in the temperature-regulated Ptac system was about 35% of total cellular protein. However, this system can not be applied to E. coli strains containing lacIq, which confers over production of the lac repressor, for high-level expression of recombinant proteins. In comparison with the lambda PL system, the Ptac-based xylanase plasmid in E. coli pop2136 gave a considerably higher specific activity of the xylanase than did the best lambda PL-based construct using the same thermal induction procedure. The high-level expression of the xylanase using the temperature-regulated Ptac system was also obtained in 10-litre fermentation studies using a fed-batch process. These results unambiguously demonstrated that the temperature-modulated Ptac system can be used for overproduction of some non-toxic recombinant proteins.
authors.affiliation: CSIRO Division of Tropical Crops and Pastures, St. Lucia, Qld, Australia.
authors.forename: G P
authors.initials: GP
authors.lastname: Xue
authors.forename: J S
authors.initials: JS
authors.lastname: Johnson
authors.forename: D J
authors.initials: DJ
authors.lastname: Smyth
authors.forename: L M
authors.initials: LM
authors.lastname: Dierens
authors.forename: X
authors.initials: X
authors.lastname: Wang
authors.forename: G D
authors.initials: GD
authors.lastname: Simpson
authors.forename: K S
authors.initials: KS
authors.lastname: Gobius
authors.forename: J H
authors.initials: JH
authors.lastname: Aylward
mesh_terms: D001483:Base Sequence; D002480:Cellulase / Q000096:biosynthesis / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005658:Fungi / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D007763:Lac Operon; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013696:Temperature; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D002480:Cellulase