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

Article Type:
Research/Original Article
Language:
English
Published:
Avicenna Journal of Medical Biotechnology, Volume:12 Issue:1, 2020
Pages:
32 - 36
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