Open AccessClonning and expression of recombinant carbapenemase KPC-2 enzyme in E. coli cytoplasm
Author(s) -
Phuong Nhat Tran,
Phuong Truc Huynh,
Trang Thi Phuong Tran,
Thuoc Linh Tran,
Van Hung Pham
Publication year - 2016
Publication title -
khoa học công nghệ
Language(s) - English
Resource type - Journals
ISSN - 1859-0128
DOI - 10.32508/stdj.v19i2.800
Subject(s) - ertapenem , klebsiella pneumoniae , recombinant dna , escherichia coli , plasmid , microbiology and biotechnology , biology , porin , expression vector , lac operon , gene , bacteria , bacterial outer membrane , biochemistry , genetics
Production of KPC-type carbapenemase is the most common carbapenem resistant mechanism in Klebsiella pneumoniae. The expression level of KPC in these strains is different and is mostly required other mechanisms to reach the higher resistant level such as porin lost or co-expression of extended spectrum β-lactamase (ESBL). To better understand the expression of KPC enzyme, the KPC-2 encoding genes from clinical isolated K. pneumoniae were cloned into pET28a plasmid. The recombinant plasmids containing of kpc-2 gene were subsequently transformed into E. coli OmniMax and were screened in kanamycine added LB media to select E. coli possessing of recombinant plasmid. Carbapenemase activity in the broth culture was checked in LB broth supplemented with 4 µg/mL of ertapenem and the expression induced with IPTG was checked by SDS-PAGE method. The results showed that this recombinant vector was capable of effective expression of KPC-2 protein in E. coli and this strain could be grown in LB broth supplemented with 4 µg/mL of ertapenem. A half of the target protein was soluble in the supernatant however it could be successfully collected from a HistrapHP affinity chromatography column. The result of this report is one of resources for further studies and applications of this KPC-2 protein in clinical research.