Sigal I S, Harwood B G, Arentzen R
Proc Natl Acad Sci U S A. 1982 Dec;79(23):7157-60. doi: 10.1073/pnas.79.23.7157.
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.
我们描述了一种方法，通过该方法将pBR322β-内酰胺酶（青霉素酶，青霉素酰胺-β-内酰胺水解酶，EC 3.5.2.6）活性位点丝氨酸-70的密码子（AGC）改变为半胱氨酸的密码子（TGC）。通过用T4 DNA聚合酶的3'至5'外切核酸酶处理pBR322的有限HgiAI消化产物，切除pBR322的相关核苷酸碱基A-G-C-A（第410-413位）。新序列T-G-C-A分两步插入。首先，将Kpn I分子接头d（T-G-G-T-A-C-C-A）连接到上述缺口处。然后用Kpn I和T4 DNA聚合酶酶切内部序列G-T-A-C，并使分子重新环化。这个编码硫醇-β-内酰胺酶的突变基因赋予大肠杆菌K-12宿主一种氨苄青霉素抗性，与pBR322赋予的抗性相比有所降低，但大于缺乏任何完整β-内酰胺酶基因的大肠杆菌的抗性。携带硫醇-β-内酰胺酶基因的大肠杆菌菌株的无细胞提取物具有对氯汞苯甲酸敏感的β-内酰胺酶活性。
