Overexpression, solubilization and refolding of a genetically engineered derivative of the penicillin-binding protein 3 of Escherichia coli K12.
Replacement of the amino-terminal 40-amino-acid region of the 588-amino-acid precursor of the membrane-bound penicillin-binding protein 3 (PBP3) by the decapeptide MKGKEFQAWI was carried out by altering the amino-coding end of the ftsI gene. Insertion of the modified gene into a runaway-replication plasmid under the control of a fused lpp promoter and lac promoter/operator, resulted in the overexpression by Escherichia coli of the modified PBP3 (designated PBP3**) in the cytoplasm. About 80% of the accumulated PBP3** underwent sequestration in the form of insoluble protein granules that were isolated by cell breakage or cell lysis. After selective removal of contaminants by an EDTA-lysozyme/DNase (deoxyribonuclease)/Nonidet extraction, treatment of the granules with guanidinium chloride followed by dialysis against buffer containing 0.5 M NaCl yielded a refolded, water-soluble PBP3**, which, upon chromatography on Superose 12, exhibited the expected 60,000 molecular mass. The refolded PBP3** bound benzylpenicillin in a 1 to 1 molar ratio, was highly sensitive to aztreonam and showed the same degree of thermostability, in terms of penicillin-binding capacity, as the parent, membrane-bound PBP3, suggesting that protein refolding occurred with formation of the correct intramolecular interactions. Two to three mg of refolded PBP3** can be obtained from 1 litre of culture of the overproducing strain.
D000217:Acyltransferases / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins; D003001:Cloning, Molecular; D003594:Cytoplasmic Granules / Q000032:analysis; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006602:Hexosyltransferases / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D002267:Muramoylpentapeptide Carboxypeptidase; D046915:Penicillin-Binding Proteins; D046928:Peptidoglycan Glycosyltransferase; D010458:Peptidyl Transferases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012995:Solubility
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C478704:FtsI protein, E coli; D006146:Guanidines; D009097:Multienzyme Complexes; D046915:Penicillin-Binding Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000217:Acyltransferases; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D046928:Peptidoglycan Glycosyltransferase; D002267:Muramoylpentapeptide Carboxypeptidase; D019791:Guanidine