Thiol-beta-lactamase: replacement of the active-site serine of RTEM beta-lactamase by a cysteine residue.

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Abstract
A procedure is described by which the codon (AGC) for the active-site serine-70 of plasmid pBR322 .beta.-lactamase (penicillinase, penicillin amido-.beta.-lactamhydrolase, EC 3.5.2.6) is altered to that for cysteine (TGC). The pertinent nucleotide bases, A-G-C-A, positions 410-413, of pBR322 are excised by treating a limited HgiAI digest of pBR322 with the 3'' .fwdarw. 5'' exonuclease of phage T4 DNA polymerase. The new sequence, T-G-C-A, is inserted in 2 steps. First, the KpnI molecular linker d(T-G-G-T-A-C-C-A) is ligated into the gap described above. The internal sequence G-T-A-C is then excised enzymatically with KpnI and T4 DNA polymerase and the molecule is recircularized. This mutant gene, which codes for a thiol-.beta.-lactamase, confers on Escherichia coli K-12 hosts an ampicillin resistance that is reduced compared with that given by pBR322 yet is greater than that of E. coli lacking any intact .beta.-lactamase gene. Cell-free extracts of E. coli strains hosting the thiol-.beta.-lactamase gene possess a p-chloromercuribenzoate-sensitive .beta.-lactamase activity.