Effect of poly-histidine tag position toward inhibition activity of secretory leukocyte protease inhibitor as candidate for material wound healing

Elly Munadziroh, Evi Umayah Ulfa, Amaliah Labiqah, One Asmarani, Ni Nyoman Tri Puspaningsih

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

Background: The Secretory Leukocyte Protease Inhibitors (SLPI) has many biological functions including anti-bacterial, anti-fungal, anti-viral, anti-inflammatory, and im-muno-modulatory. Previous studies have shown that gene-encoding human SLPI have successfully been expressed in Escherichia coli (E. coli) with a C-terminal poly-histidine 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.

Original languageEnglish
Pages (from-to)32-36
Number of pages5
JournalAvicenna Journal of Medical Biotechnology
Volume12
Issue number1
Publication statusPublished - 2020

Keywords

  • Escherichia coli
  • Gene expression
  • Poly histidine
  • Secretory leukocyte protease inhibitor

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