Method for cloning and expression of BsrFI restriction endonucle

Details Method for cloning and expression of BsrFI restriction endonucle Method for cloning and expression of BsrFI restriction endonucle

1999-05-07

2000-05-23

Patterson, Jr., Charles L.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

4352523, 4353261, 536 232, C12N 922, C12N 1555

Patent

active

060664878

ABSTRACT:
BsrFI restriction enzyme was purified from Bacillus stearothermophilus to near homogeneity. The protein was sequenced to obtain its N-terminus amino acid sequence. A set of denegerate primers were synthesized based on the aa sequence. The first 18 codons encoding BsrFI restriction endonuclease (bsrFIR) was amplified by PCR and its coding sequence was obtained. The methylase selection method was used to clone BsrFI methylase gene (bsrFIM). Two clones were found to be resistant to BsrFI digstion. The entire insert in one clone was sequenced and the insert encodes the BsrFI methylase (M. BsrFI). In addition, a small truncated open reading frame adjacent to the methylase gene has homology to Cfr10I restriciton endonuclease in a BlastX homology search in Genbank database. BsrFI and Cfr10I are isoschizomer that recognizes and cleaves 5'R CCGGY3'. Two primers were used to amplify the bsrFIR gene, The forward primer is a degenerate primer designed from the N-terminus aa sequence and the reverse primer is the bona fide sequence derived from the BsrFI methylase.sup.+ clone. The bsrFIR gene was amplified by PCR, ligated into a T7 expression vector pET21at and the ligated DNA was transformed into premodified cells ER2566 [pLG339-BsrFIM]. The final expression strain is ER2566 [pLG339-BsrFIM, pET21at-BsrFIR]. Recombinant BsrFI activity was detected in E. coli cell extract. BsrFI is cloned from a thermophile Bacillus stearothermophilus. Thus, BsrFI a thermostable enzyme and it is active at 37.degree. C. to 65.degree. C.

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