Expression of Recombinant pET22b-LysK-Cysteine/Histidine-Dependent Amidohydrolase/Peptidase Bacteriophage Therapeutic Protein in Escherichia coli BL21 (DE3)

Haddad-Kashani, H. and Moniri, R. (2015) Expression of Recombinant pET22b-LysK-Cysteine/Histidine-Dependent Amidohydrolase/Peptidase Bacteriophage Therapeutic Protein in Escherichia coli BL21 (DE3). Osong Public Health and Research Perspectives, 6 (4). pp. 256-260.

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Objectives: Bacteriophage-encoded endolysins are a group of enzymes that act by digesting the peptidoglycan of bacterial cell walls. LysK has been reported to lyse live staphylococcal cultures. LysK proteins containing only the cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) domain has the capability to show lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus (MRSA). The aim of this study was to clone and express LysK-CHAP domain in Escherichia coli BL21 (DE3) using pET22b as a secretion vector. The pET22b plasmid was used, which encoded a pelB secretion signal under the control of the strong bacteriophage T7 promoter. Methods: The E. coli cloning strains DH5α and BL21 (DE3) were grown at 37°C with aeration in the Luria-Bertani medium. A plasmid encoding LysK-CHAP in a pET22b backbone was constructed. The pET22b vector containing LysK-CHAP sequences were digested with NcoI and HindIII restriction enzymes. Cloning accuracy was confirmed by electrophoresis. The pET22b-LysK plasmid was used to transform the E. coli strain BL21. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1mM to induce T7 RNA polymerase-based expression. Finally, western blot confirmed the expression of target protein. Results: In this study, after double digestion of pEX and pET22b vectors with HindIII and NcoI, LysK gene was cloned into two HindIII and NcoI sites in pET22b vector, and then transformed to E. coli DH5α. Cloning was confirmed with double digestion and analyzed with agarose gel. The recombinant pET22b-LysK plasmid was transformed to E. coli BL21 and the expression was induced by IPTG. The expression was confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting method. Observation of a 28.5 kDa band confirmed LysK protein expression. Conclusion: In the present study, LysK-CHAP domain was successfully cloned and expressed at the pET22b vector and E. coli BL21 (DE3). © 2015.

Item Type: Article
Additional Information: cited By 1
Uncontrolled Keywords: amidase; bacterial protein; cysteine; histidine; isopropyl thiogalactoside; LysK protein; peptidase; restriction endonuclease; RNA polymerase; unclassified drug, Article; bacterial gene; bacterial strain; bacteriophage; bacterium isolate; controlled study; electrophoresis; Enterobacteria phage T7; Escherichia coli; Escherichia coli BL21; gene sequence; HindIII gene; LysK gene; methicillin resistant Staphylococcus aureus; molecular cloning; NcoI gene; nonhuman; polyacrylamide gel electrophoresis; priority journal; protein domain; protein expression; Staphylococcus; Western blotting
Subjects: Immunology and Microbiology
Divisions: Faculty of Medicine > Basic Sciences > Department of Microbiology & Immunology
Depositing User: editor . 2
Date Deposited: 08 Mar 2017 13:49
Last Modified: 08 Mar 2017 13:49

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