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재조합 베타갈락토시다제 - 프리 S2 융합단백질에 존재하는 두 개의 메치오닌 위치에 올리고뉴클레오티드를 이용한 변이유도
박종광,이상철,최영철,한문희,유명희 ( Chong Kwnag Park,Sang Chul Lee,Young Chul Choi,Moon H . Han,Myeong Hee Yu ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.1
E. coli JM109 cells harboring pCTHB20 plasmid overproduced β-galactosidase-preS2 (βgal-preS2) fusion proteins with a yield of 50% of total cellular proteins (Choi et al., 1988). In order to facilitate the isolation of preS2 peptide out of the fusion protein after CNBr cleavage, the lacZ sequence was rearranged to construct pCTHB30 plasmid. Two internal methionine codons in the lacZ region of pCTHB20 and pCTHB30 was also substituted with valine codons by oligonucleotide-directed mutagenesis. The EcoRI-XbaI restriction fragment of lacZ was isolated from the plasmid pCTHB20 and was inserted into multicloning site of M13 mp18 vector. Two synthetic complementary oligonucleotides (17-mer each) with desired nucleotide changes were used simultaneously as primers in synthesizing a complementary strand. Mutant clones were screened by DNA hybridization with probes of ^(32)P-labeled mutagenic oligonucleotides, and the nucleotide substitutions were confirmed by DNA sequencing. E. coli JM109 cells transformed with pCMHB20 and pCMHB30 in which the lacZ regions were substituted with the mutant counterpart produced βgal-preS2 fusion proteins as efficiently as those with pCTHB20 and pCTHB30.