Hermansyah, Hermansyah (2018) Cloning of SLPI gene containing HM-1 signal peptide in Saccharomycees cerevisiae (Similirity). Unsri, Unsri. (Unpublished)
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Abstract
SLPI is a non-glycosylated protein and it composed of 107 amino acid secreted by epithelial cells, macrophages, and neutrophils. This protein has multiple functions i.e. anti-inflammatory, anti-microbial, and wound healing. SLPI is involved in wound healing by protecting epithelial tissues from serine protease degradation during inflammation. Our previous study has successfully cloned SLPI gene generated from the amniotic membrane into the pET-101/D-TOPO vector. However, E. coli is not a safe host for recombinant protein production because its cell wall contains toxic pyrogens. To overcome the limitation, we cloned SLPI gene into the pYHM1 vector for expression in Saccharomyces cerevisiae. The American Food and Drug Administration (FDA) categorized S. cerevisiae as a Generally Recognized as Safe (GRAS) microorganism. The pYHM1 vector contains HM1 signal peptide to mediate protein secretion. SLPI gene from amniotic membrane was amplified from pET-ESLPI. The PCR product was digested by SacI and EcoRI and cloned into a pYHM1 vector to produce pY_SLPI recombinant plasmid in E. coli TOP10. The recombinant plasmid was then transformed into S. cerevisiae BJ1824 using lithium acetate method. The SLPI gene was successfully amplified with size ~339 bp and cloned into the pYHM1 vector in E. coli TOP10. The restriction and nucleotide sequence analysis showed that transformant number 31 contained SLPI gene. The pY_SLPI recombinant plasmid has been successfully transformed into S. cerevisiae BJ1824. SLPI recombinant was detected in intracellular and cell-associated by SDS PAGE.
| Item Type: | Other |
|---|---|
| Subjects: | Q Science > QD Chemistry > QD415-436 Biochemistry |
| Divisions: | 08-Faculty of Mathematics and Natural Science > 47201-Chemistry (S1) |
| Depositing User: | Hermansyah Hermansyah |
| Date Deposited: | 29 Aug 2020 12:41 |
| Last Modified: | 29 Aug 2020 12:41 |
| URI: | http://repository.unsri.ac.id/id/eprint/34095 |
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