Konstruksi Vektor Ekspresi Gen KEX2-620 dengan Metode Sub-Kloning pada pPICZaB

Abstract

Gen KEX2 menyandi enzim Kex2 (Kexin, EC3.4.21.61), endopeptidase yang termasuk famili substilisin dari serine proteinase. Enzim ini memotong protein secara spesifik pada ikatan karboksilat setelah dua asam amino basa berurutan, yaitu setelah -Lys-Arg/X atau -Arg-Arg/X. Kex2 umumnya ditemukan pada jalur sekretori khamir seperti Saccharomyces cerevisiae dan Komagataella phaffii, berperan penting dalam sekresi protein. Penelitian ini bertujuan melakukan konstruksi vektor ekspresi gen KEX2-620 dengan metode sub-kloning ke dalam plasmid pPICZaB. Gen KEX2-620 yang merupakan varian gen KEX2 tanpa domain transmembran dan domain Ser/Thr, dikonstruksi dengan teknik PCR dari plasmid pD902-Kex2-699. Amplifikasi gen KEX2-620 berhasil dilakukan dan diperoleh fragmen gen berukuran 1560 bp. Simpulan penelitian ini adalah plasmid rekombinan pPICZaB-Kex2-620 berukuran 5085 bp berhasil ditransformasi ke dalam Escherichia coli strain DH5a, tetapi berdasarkan hasil verifikasi PCR, masih ada plasmid pPICZaB yang membentuk self-ligation.

The KEX2 gene encodes an enzyme, Kex2 (Kexin, EC3.4.21.61), which is an endopeptidase belonging to the subtilisin family of serine proteinases. This enzyme cleaves proteins specifically at the carboxylic bond after two consecutive basic amino acids, namely after -Lys-Arg/X or -Arg-Arg/X. Kex2 is commonly found in the secretory pathway of yeasts such as Saccharomyces cerevisiae and Komagataella phaffii, and play a crucial role in protein secretion. This research aims to construct the KEX2-620 gene expression vector by sub-cloning into the pPICZaB plasmid. The KEX2-620 gene, which is a variant of the KEX2 gene without the transmembrane domain and Ser/Thr domain, was amplified using the PCR technique from plasmid pD902-Kex2-699. Amplification of the KEX2-620 gene was successfully carried out and a 1560 bp gene fragment was obtained. The conclusion of this study is that the 5085 bp recombinant plasmid pPICZaB-Kex2-620 was successfully transformed into Escherichia coli strain DH5a, but based on PCR verification results, there was still self-ligation of the pPICZaB plasmid.