Oligonucleotide-directed mutagenesis of two methionine residues in recombinant beta-galactosidase-preS2 fusion proteins

Cited 0 time in scopus
Metadata Downloads
Title
Oligonucleotide-directed mutagenesis of two methionine residues in recombinant beta-galactosidase-preS2 fusion proteins
Author(s)
Chong Kwang Park; Sang Chul Lee; Young Chul Choi; Moon Hi Han; Myeong Hee Yu
Bibliographic Citation
Korean Biochemical Journal, vol. 22, no. 1, pp. 38-44
Publication Year
1989
Abstract
E. coll JM109 cells harboring pCTHB20 plasmid overproduced ^-galactosidase-preS2 (/9gal-preS2) fusion proteins with a yield of 50% of total cellular proteins (Choi et al., 1988). In order to facilitate the isolation ofpreS2 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-Xbal restriction fragment of lacZ was isolated from the plasmid pCTHB20 and was inserted into multicloning site of M13 mpl8 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 32P-labeled mutagenic oligonucleotides, and the nucleotide substitutions were confirmed by DNA sequencing. E. coll JM109 cells transformed with pCMHB20 and pCMHB30 in which the lacZ regions were substituted with the mutant counterpart produced 단 gal-preS2 fusion proteins as efficiently as those with pCTHB20 and pCTHB30.
ISSN
0368-4881
Publisher
Korea Soc-Assoc-Inst
Type
Article
Appears in Collections:
Division of Biomedical Research > Metabolic Regulation Research Center > 1. Journal Articles
Files in This Item:
  • There are no files associated with this item.


Items in OpenAccess@KRIBB are protected by copyright, with all rights reserved, unless otherwise indicated.