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Overexpression, solubilization and refolding of a genetically engineered derivative of the penicillin‐binding protein 3 of Escherichia coli K12
Author(s) -
Belder J. BartholoméDe,
NguyenDisteche M.,
HoubaHerin N.,
Ghuysen J. M.,
Maruyama I. N.,
Hara H.,
Hirota Y.,
Inouye M.
Publication year - 1988
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1988.tb00058.x
Subject(s) - escherichia coli , biochemistry , biology , penicillin binding proteins , lysis , plasmid , microbiology and biotechnology , lysozyme , amino acid , gene
Summary Replacement of the amino‐terminal 40‐amino‐acid region of the 588‐ammo‐acid precursor of the membrane‐bound penicillin‐binding protein 3 (P6P3) by the decapeptide MKGKEFQAWI was carried out by altering the amino‐coding end of the ftsl gene. Insertion of the modified gene into a runaway‐replication plasmid under the control of a fused Ipp 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 (de‐oxyribonuclease)/Nonidet extraction, treatment of the granules with guanidinium chloride followed by dialysis against buffer containing 0.5 M NaCI yielded a refolded, water‐soluble PBP3**, which, upon chromatography on Superose 12, exhibited the expected 60000 molecular mass. The refolded PBP3** bound benzytpenicillin 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 PBP** can be obtained from 1 litre of culture of the overproducing strain.