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

en 32153736 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 N-succinyil-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 C-terminal 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. Escherichia coli (E. coli), Gene expression, Poly histidine, Secretory leukocyte protease inhibitor 32 36 https://www.ajmb.org/En/Article.aspx?id=20413 https://www.ajmb.org/PDF/En/FullText/20413.pdf EllyMunadzirohDepartment of Dental Material and Technology, Faculty of Dentistry, Universitas Airlangga, Surabaya, Indonesia31550Evi UlfaDepartment of Chemistry, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia31551AmaliahLabiqahDepartment of Health Analysis, Stikes Kesetiakawanan Sosial Indonesia, Jakarta, Indonesia31552OneAsmaraniProteomic Study Group, Institute of Tropical Disease, Universitas Airlangga, Surabaya, Indonesia31553Ni NyomanPuspaningsihProteomic Study Group, Institute of Tropical Disease, Universitas Airlangga, Surabaya, Indonesia31554