Avicenna J Med Biotech arij002 Avicenna Journal of Medical Biotechnology 2008-2835 2008-4625 Avicenna Research Institute ajmb20413 Effect of Poly-Histidine Tag Position toward Inhibition Activity of Secretory Leukocyte Protease Inhibitor as Candidate for Material Wound Healing MunadzirohEllyUlfaEvi LabiqahAmaliahAsmaraniOnePuspaningsihNi Nyoman 12 1 32 36 9 1 2019 22 4 2019

<p style="text-align:justify"><span style="font-size:9.5pt">Background:</span> 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 <em>Escherichia coli (E. coli)</em> 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.<span style="font-size:2.0pt">&nbsp;</span></p> <p style="text-align:justify"><span style="font-size:9.5pt">Methods:</span> 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 <em>E. coli </em>TOP10. Recombinant plasmids were verified by using restriction analysis and nucleotide sequence analysis. pET-NSLPI and pET-CSLPI were then subcloned in <em>E. coli</em> BL21(DE3) for its expression. The SLPI protein was expressed using Isopropyl &beta;-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.<span style="font-size:2.0pt">&nbsp;</span></p> <p style="text-align:justify"><span style="font-size:9.5pt">Results:</span> The SLPI gene was successfully cloned and expressed in <em>E. coli</em> 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%.<span style="font-size:2.0pt">&nbsp;</span></p> <p><span style="font-size:9.5pt">Conclusion:</span> The His-tag position on the C-terminal of SLPI reduced the inhibition activity of SLPI.</p>