Purification of Antibacterial CHAPK Protein Using a Self-Cleaving Fusion Tag and Its Activity Against Methicillin-Resistant Staphylococcus aureus

Abstract

Therapeutic LysK-CHAP is a potent anti-staphylococcal protein that could be utilized as an antibiotic substitute. Intein-mediated protein purification is a reasonable and cost-effective method that is most recently used for recombinant therapeutic protein production. Intein (INT) is the internal parts of the protein that can be separated from the immature protein during protein splicing process. This sequence requires no specific enzyme or cofactor for separation. INT sequence and their characteristic of self-cleavage by thiol induction, temperature, and pH changes are used for protein purification. The current study presents the expression of CHAP_K262 domain of LysK gene that is fused with INT/chitin-binding sequence while evaluating its purification procedure and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The coding gene sequence of LysK-CHAP (CHAP_K262) in pET22-b was amplified with polymerase chain reaction (PCR); the digested product was then cloned into the pTXB1 vector. Electrophoresis confirmed the cloning accuracy of the gene. The pTXB1-CHAP_K262 plasmid was transformed to the Escherichia coli ER₂₅₆₆ (E. coli ER₂₅₆₆) expression strain and analyzed for expression of the recombinant protein by SDS-PAGE and Western blotting methods. Finally, CHAP_K262 was purified by chitin affinity column using INT tag technology and confirmed by SDS-PAGE. Lytic activity of the purified protein was investigated by disk diffusion method. Cloning of CHAP_K262 into the pTXB1 vector, which comprised INT/chitin-binding sequence, was successfully achieved. The SDS-PAGE data also revealed successful expression of the CHAP_K262-INT fusion protein and Western blotting method validated the accuracy of the protein. Moreover, purification of CHAP_K262 protein was induced by dithiothreitol (DTT) and confirmed by SDS-PAGE. Finally, inhibition zone in MRAS culture medium confirmed antibacterial activity of the protein. Application of intein-mediated antibacterial protein is an appropriate and streamlined method for one-step purification of CHAP_K262 as a therapeutic and antibacterial protein. Self-cleaving tags like intein are cost-effective and could be used as a proper purification method for industrial purposes.

Keywords: Antibacterial protein; CHAPK262 domain; Expression; Purification; Self-cleaving tag; Staphylococcus aureus.