Localization and characterization of inclusion bodies in recombinant Escherichia coli cells overproducing penicillin G acylase.
Various concentrations of isopropyl beta-D-thiogalactopyranoside (IPTG) were used to induce production of the enzyme penicillin G acylase by recombinant Escherichia coli harboring plasmid pQEA11. The plasmid pQEA11 carries a wild-type pga gene, which is under the control of the tac promoter and lacIq. At low IPTG concentrations (0.025-0.1 mM), enzyme activity increased with increasing IPTG concentrations. At higher IPTG concentrations (0.2 and 0.5 mM), enzyme activity declined progressively. Examination of induced recombinant E. coli cells by transmission electron microscopy showed the presence of only periplasmic inclusion bodies at low IPTG concentrations (up to 0.1 mM) and both periplasmic and cytoplasmic inclusion bodies at high IPTG concentrations (0.2 mM and 0.5 mM). Results from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblots of whole-cell proteins, membrane proteins and inclusion body proteins in these cells indicated that cytoplasmic inclusion bodies constituted an accumulation of preproenzyme (i.e., precursor polypeptide containing a signal peptide) and that periplasmic inclusion bodies constituted an accumulation of proenzyme (i.e., precursor polypeptide lacking a signal peptide).
Sriubolmas
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N
Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand.
Panbangred
W
W
Sriurairatana
S
S
Meevootisom
V
V
D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D015151:Immunoblotting; D002479:Inclusion Bodies / Q000187:drug effects / Q000648:ultrastructure; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D056968:Lac Repressors; D008565:Membrane Proteins / Q000032:analysis; D008854:Microscopy, Electron; D010399:Penicillin Amidase / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000032:analysis / Q000502:physiology; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000235:genetics; D015183:Restriction Mapping
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D012097:Repressor Proteins; D007544:Isopropyl Thiogalactoside; D010399:Penicillin Amidase