EVI UMAYAH ULFA, 081517027306 (2019) KONSTRUKSI GEN PENYANDI SECRETORY LEUKOCYTE PROTEASE INHIBITOR (SLPI) DAN PROTEIN DISULFIDA ISOMERASE (PDI) PADA INANG Saccharomyces cerevisiae BJ1824. Disertasi thesis, Universitas Airlangga.

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Abstract

Secretory Leukocyte Protease Inhibitor (SLPI) is an elastase inhibitor that can be used as a wound healing, anti-inflammatory, antibacterial, antifungal and antiretroviral. In the wound healing process, SLPI prevents tissue degradation due to excessive product of neutrophils and suppresses the expression of various cytokines such as IL8 and IL10 which will stimulate tissue damage through inhibition of NF transcription factors. In line with the advances of science and technology including biotechnology, proteins that play an important role are produced by genetic engineering techniques to obtain recombinant proteins and study their molecular characteristics. S. cerevisiae is a safe host and contains no lipopolysaccharide pyrogen as in Eschericia coli. This host is also capable to produce heterologous proteins doing the post-translation modification and can be secreted to the media. The secreted protein is a better option because of the lack of host protein contamination. In this study, the construction of the SLPI gene was done by (1). fusing with HM1 signal peptides for secretion and (2). co-expression with the PDI1 gene to increase recombinant SLPI expression. SLPI is a protein that has eight disulfide bonds. Protein Disulfide Isomerase (PDI) has a role in catalyzing the formation of disulfide bonds. Increasing number of PDI in the cell is expected to assist the folding process of the SLPI as well as increasing the number of secreted SLPIs. The construction of SLPI gene co-expression with the PDI1 gene was carried out using the pAT425 vector expression which has two multiple cloning sites (MCS). The SLPI gene isolated from the Indonesian amniotic membrane. Meanwhile, the plasmid used for the pAT425 coexpression system and the signal peptide used for SLPI secretion comes from the HM1 killer toxin. The purpose of this study is to gain an SLPI secretion system in S. cerevisiae hosts and SLPI recombinant proteins which acts as elastase inhibitors. Steps of the study include: 1) cloning of the SLPI and PDI1 genes using the pAT425 co-expression vector, 2) SLPI expression and secretion analysis in S. cerevisiae hosts, and 3) activity test of in vitro elastase inhibitory. The construction of the SLPI and PDI1 gene co-expression system was carried out in four stages. Firstly, the SLPI gene was fused with the HM1 signal peptide coding gene. Fusion was carried out by inserting the SLPI gene into the pYHM1 vector. Amniotic membrane SLPI gene was amplified by PCR using pFSLPISacI and pRSLPIEcoRIprimers.The amplicon and pYHM1 vector were digested using the SacI/ EcoRI and transformed into E. coli TOP10 after being ligated for 24-48 hours at 4° C. That recombinant plasmid is called pY_SLPI. Secondly, the fusion of SLPI gene and HM1 signal peptide (hmSLPI) was subcloned into the pAT425 plasmid on MCS-1. Fusion of the hmSLPI gene was isolated from the pY_SLPI recombinant plasmid by PCR using primers pFhmSLPIPmeI, pRhmSLPIAvrII. Amplicons and pAT425 vectors were ligated for 24-48 hours at 4C, after each digested with the PmeI/AvrII. The ligation results were transformed into E.coli TOP10. That recombinant plasmid is called pAT_hmSLPI and subsequently transformed into S. cerevisiae BJ1824 to determine SLPI expression without the addition of PDI1 gene. Third, cloning of the PDI1 gene into pAT425 vector in MCS2 by amplifying the PDI1 gene from the BYP7700 plasmid. PCR was performed using pFPDIMluI and pRPDINotI primers. PCR results were digested with MluI and NotI and ligated with pAT425 which had previously been digested with the same restriction enzyme of MluI/NotI. Ligation results were transformed into E. coli TOP10. The recombinant plasmid obtained is calledpAT_PDI. The fourth step was cloninghmSLPI gene fusion in the recombinant plasmid of pAT_PDI. The procedure was done as in the second step. Yet the plasmid was pAT_PDI. The ligation results of the hmSLPI and pAT_PDI genes, each of which was digested with the PmeI/AvrII enzymes, were transformed into E.coli TOP10. This recombinant plasmid was called pAT_PDI_hmSLPI and was then sub-cloned into S. cerevisiae BJ1824 to find out the SLPI expression. Analysis of expression and secretion was carried out for 3 days using liquid YPD rich media and liquid YNBD selective media. Yeast cell pellets were separated from culture media by centrifugation. The extracellular protein fraction was obtained by concentrating the culture medium using a 10 kDa MWCO membrane.Meanwhile, the intracellular protein fraction was obtained by breaking the yeast using a glass bead for 10 minutes with 30 seconds on and 30 seconds off. Proteins attached to the cell membrane (associated cells) were also analyzed for expression by dissolving lysis pellets with 0.2 M tris-HCl buffer pH 8. Characterization of recombinant SLPI molecular weight was determined by using SDS-PAGE and Western Blot. SLPI levels were determined using ELISA. The intracellular and extracellular protein fraction was determined by using the serine protease inhibition activity. The SLPI inhibitory activity test of chymotrypsintype protease serine was carried out by determining a decrease in the amount of pnitroaniline (pNA) due to the inhibition of chymotrypsin activity against the specific substrate of N-suc-Ala-Ala-Pro-Phe-ρ-nitroanilide (NPN). In this study, SLPI secretion vector was obtained without the addition of PDI1 gene (pAT_hmSLPI) and by adding PDI1 gene (pAT_PDI_hmSLPI) in S. cerevisiae BJ1824. The SLPI gene carries HM1 signal peptides for the purpose of secreting out yeast cells. The hmSLPI fusion gene and PDI1 gene were successfully amplified at anealing temperature of 59.3 °C by using pY_SLPI and BYP7700 plasmids as a template, respectively. The results of restriction analysis, PCR, and sequencing confirmed that hmSLPI fusion gene and PDI1 gene were successfully cloned into pAT425 plasmids. Recombinant plasmid of pAT_hmSLPI, pAT_PDI and pAT_PDI_hmSLPI were successfully subcloned into S. cerevisiae BJ1824. The characterization of recombinant SLPI using SDS-PAGE and Western blot indicated that SLPI was successfully expressed as a fusion protein with HM1 signal peptide with a size of ~ 16.3 kDa. The concentration of SLPI levels in intracellular fraction without and with the addition of PDI produced in YPD rich media were 4.4 ± 0.069 ng/mL and 1.98 ± 0.006 ng/mL, respectively. The concentration of SLPI that were cells associated without and with the addition of PDI were 2.41 ± 0.004 ng/mL and 3.74 ± 0.005 ng/mL while the SLPI level in extracellular fraction without and with the addition of PDI were 0.29 ± 0.017 ng/mL and 0.317± 0.03 ng/mL. Extracellular protein fraction using SDS PAGE showed no SLPI band. Inhibition percentage of chymotrypsin between SLPI with and without the addition of PDI produced in YPD rich media were 50.19 ± 12.89 and 70.59 ± 8.47 respectively. Meanwhile, the inhibition percentage in YNBD selective media was 21.78 ± 0.87 % and 46.64 ± 2.31%. The addition of gene encoding PDI could increase SLPI expression and SLPI inhibitory activity against chymotrypsin. Inhibitory activity was only confirmed by the extracellular protein fraction. The absence of SLPI band on SDS PAGE, the presence of inhibitory activity of the extracellular protein fraction and SLPI using ELISA showed that SLPI was successfully secreted from yeast cells. These results indicated that the HM1 signal peptide is capable of secreting SLPI even in small amounts. Most of the expressed SLPIs were in cells or attached to yeast cell membranes. The result of this study have been published on AIP conference proceedings and the Avicenna Journal of Medical Biotechnology.

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