دورية
Thiol-beta-lactamase: replacement of the active-site serine of RTEM beta-lactamase by a cysteine residue.
| العنوان: | Thiol-beta-lactamase: replacement of the active-site serine of RTEM beta-lactamase by a cysteine residue. |
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| المؤلفون: | Sigal, I S, Harwood, B G, Arentzen, R |
| المصدر: | Proceedings of the National Academy of Sciences of the United States of America; December 1982, Vol. 79 Issue: 23 p7157-7160, 4p |
| مستخلص: | We describe a procedure by which the codon (AGC) for the active-site serine-70 of 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' leads to 5' exonuclease of T4 DNA polymerase. The new sequence, T-G-C-A, is inserted in two steps. First, the Kpn I 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 Kpn I 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. |
| قاعدة البيانات: | Supplemental Index |
| تدمد: | 00278424 10916490 |
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| DOI: | 10.1073/pnas.79.23.7157 |