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Characterization of synthetic gene encoding Secretory Leucocyte Protease Inhibitor cloned in Escherichia coli host cell to improve inhibition abilityas a candidate of material wound healing
Elly Munadziroh1.
Introduction: Secretory Leukocyte Protease Inhibitor (SLPI) is a protein involved in tissue repair and oral wound healing processes. The gene encoding SLPI was included from amniotic membrane. SLPI is a potent anti-protease, anti-inflammatory, bactericidal, antifungal, and tissue repair agent. It is a non-glycosylated protein of 11.7 kDa and stable in acidic conditions. It has an isoelectric point of more than 9.5. The purpose of this study was to construct a SLPI synthetic gene, and to clone it in Escherichia coli for its expression in order to get the optimal expression of recombinant protein of SLPI. Methods: The synthetic gene encoding SLPI was designed and commercially synthe-sized by Genscript in pUC57. This gene was amplified using PCR, then ligated into pET-30a plasmid after digestion with KpnI and XhoI. Ligated mixtures were transformed into E. coli TOP10 and the plasmid-containing cells were selected using LB/kanamycine plates. The recombinant plasmid (pET/SLPIopt) was verified using restriction analysis and nucleotide sequence analysis. pET/SLPIopt was then subcloned in E. coli BL21(DE3) for its expression. The SLPI protein was expressed by using IPTG induction. Results: The synthetic gene encoding SLPI has been successfully constructed in the size of 412 bp and inserted into pET 30a in E. coli TOP10. The expression plasmid (pET/SLPIopt) has been successfully sub-cloned into E.coli BL21(DE3). The expression result showed that SLPI could be produced by using 25, 50, 100, 500, μM IPTG and mostly accumulated in cell pellet. Conclusion: The SLPI synthetic gene cloned in Escherichia coli can express the optimal recombinant protein of SLPI.
Affiliation:
- Universitas Airlangga, Indonesia
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Indexation |
Indexed by |
MyJurnal (2021) |
H-Index
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3 |
Immediacy Index
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0.000 |
Rank |
0 |
Indexed by |
Scopus 2020 |
Impact Factor
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CiteScore (0.2) |
Rank |
Q4 (Medicine (all)) |
Additional Information |
SJR (0.144) |
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