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

Overview

Journal Avicenna J Med Biotechnol

Specialty Biomedical Engineering

Date 2020 Mar 11

PMID 32153736

Citations 1

Authors

Elly Munadziroh

Evi Umayah Ulfa

Amaliah Labiqah

One Asmarani

Ni Nyoman Tri Puspaningsih

Affiliations

Soon will be listed here.

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 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 TOP10. Recombinant plasmids were verified by using restriction analysis and nucleotide sequence analysis. pET-NSLPI and pET-CSLPI were then subcloned in 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 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.

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