Characterization of the T7 promoter system for expressing penicillin acylase in Escherichia coli.
The pac gene encoding penicillin acylase (PAC) was overexpressed under the regulation of the T7 promoter in Escherichia coli. PAC, with its complex formation mechanism, serves as a unique target protein for demonstration of several key strategies for enhancing recombinant protein production. The current T7 system for pac overexpression was fraught with various technical hurdles. Upon the induction with a conventional inducer of isopropyl-beta-D-thiogalactopyranoside (IPTG), the production of PAC was limited by the accumulation of PAC precursors (proPAC) as inclusion bodies and various negative cellular responses such as growth inhibition and cell lysis. The expression performance could be improved by the coexpression of degP encoding a periplasmic protein with protease and chaperone activities. In addition to IPTG, arabinose was shown to be another effective inducer. Interestingly, arabinose not only induced the current T7 promoter system for pac expression but also facilitated the posttranslational processing of proPAC for maturation, resulting in significant enhancement for the production of PAC. Glycerol appeared to have an effect similar to, but not as significant as, arabinose for enhancing the production of PAC. The study highlights the importance of developing suitable genetically engineered strains with culture conditions for enhancing recombinant protein production in E. coli.
Xu
Yali
Y
Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, N2L 3G1 Canada.
Rosenkranz
Stefan
S
Weng
Chiao-Ling
CL
Scharer
Jeno M
JM
Moo-Young
Murray
M
Chou
C Perry
CP
D001089:Arabinose / Q000378:metabolism; D017123:Bacteriophage T7 / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005990:Glycerol / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D002479:Inclusion Bodies / Q000378:metabolism; D007218:Industrial Microbiology; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D010399:Penicillin Amidase / Q000096:biosynthesis / Q000235:genetics*; D033903:Periplasmic Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012697:Serine Endopeptidases / Q000378:metabolism
D006360:Heat-Shock Proteins; D033903:Periplasmic Proteins; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D001089:Arabinose; C061682:DegP protease; D012697:Serine Endopeptidases; D010399:Penicillin Amidase; D005990:Glycerol