IADR Abstract Archives
Expression of Synthetic Gene Encoding Secretory Leucocyte Protease Inhibitor Cloned on pET-30a plasmid in Escherichia coli Host Cell
Objectives: This research to construct 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 synthesized by Genscript in pUC57. This gene was amplified using PCR, then ligated into pET-30a plasmid after digested with KpnI and XhoI. Ligated mixtures were transformed into E. coli TOP10 and the plasmid containing cell were selected on LB/kanamycine plates. Recombinant plasmid (pET/SLPIopt) was verified by 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 succesfully constructed in the size of 412 bp and inserted into pET 30a in E. coli TOP10. The expression plasmid (pET/SLPIopt) has been succesfully sub-cloned into E.coli BL21(DE3). The expression result showed that SLPI could be produced by using 25, 50, 100, 500, mM IPTG and mostly accumulated in cell pellet.
Conclusions: SLPI synthetic gene that cloned in Escherichia coli can express the optimal recombinant protein of SLPI.
Methods: The synthetic gene encoding SLPI was designed and commercially synthesized by Genscript in pUC57. This gene was amplified using PCR, then ligated into pET-30a plasmid after digested with KpnI and XhoI. Ligated mixtures were transformed into E. coli TOP10 and the plasmid containing cell were selected on LB/kanamycine plates. Recombinant plasmid (pET/SLPIopt) was verified by 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 succesfully constructed in the size of 412 bp and inserted into pET 30a in E. coli TOP10. The expression plasmid (pET/SLPIopt) has been succesfully sub-cloned into E.coli BL21(DE3). The expression result showed that SLPI could be produced by using 25, 50, 100, 500, mM IPTG and mostly accumulated in cell pellet.
Conclusions: SLPI synthetic gene that cloned in Escherichia coli can express the optimal recombinant protein of SLPI.