Effect of Poly-Histidine Tag Position toward Inhibition Activity of Secretory Leukocyte Protease Inhibitor as Candidate for Material Wound Healing

Background: the secretory leukocyte protease inhibitors (slpi) has many biological functions including anti-bacterial, anti-fungal, anti-viral, anti-inflammatory, and immuno- modulatory. previous studies have shown that gene-encoding human slpi have successfully been expressed in escherichia coli (e. coli) with a c-terminal polyhistidine tag (his-tag). the aim of this research was to investigate the inhibition activity of n-terminal his-tag position (nslpi) and c-terminal his-tag position (cslpi). we hypothesized that a his-tag close to an active site slpi domain may interfere with the inhibition activity of slpis. methods: a nslpi and cslpi were constructed with polymerase chain reaction (pcr) amplification. the pcr products were then ligated into pet-30a plasmid and transformed into e. coli top10. recombinant plasmids were verified by using restriction analysis and nucleotide sequence analysis. pet-nslpi and pet-cslpi were then subcloned in e. coli bl21(de3) for its expression. the slpi protein was expressed using isopropyl β-d-1-thiogalactopyranoside induction (iptg). the inhibition effect of both slpi against porcine pancreatic elastase (ppe) enzyme was tested using the nsuccinyil- alanyl-l-alanyl-l-prolyl-l-phenylalanyl-4-nitroanalide (npn) substrate. results: the slpi gene was successfully cloned and expressed in e. coli bl21. fusion proteins of nslpi and cslpi were generated with his-tag in the n-terminal and cterminal position, respectively. the inhibition effect of nslpi and cslpi on ppe indicated that both slpi were active. the inhibition activity of nslpi was 66.7%, higher than cslpi by 44.4%. conclusion: the his-tag position on the c-terminal of slpi reduced the inhibition activity of slpi.