Construction of Saccharomyces cerevisiae KEX2-650 gene expression vector and its introduction into Escherichia coli DH5?

Abstract

Abstract. Adibah DI, Fuad AM, Budiarti S, Pratiwi RD. 2022. Construction of Saccharomyces cerevisiae KEX2-650 gene expression vector and its introduction into Escherichia coli DH5?. Biodiversitas 23: 4289-4296. Kex2 (EC 3.4.21.61) is a serine protease that binds Ca²⁺ molecules and naturally found intracellularly as a transmembrane protein. Kex2 has a unique function, the conversion process of recombinant protein precursor into mature protein. Kex2 from Saccharomyces cerevisiae has similar functions also with furin in mammals (about 47% of sequence similarity). To gain advantage, extracellular Kex2 would be highly favorable for this process. This study aimed to construct recombinant Kex2 that could be produced extracellularly in Pichia pastoris host through pD902-KEX2-699 vector (synthetic) with FLAG-tag and 6 His-tag by removing most of C-terminal region, including transmembrane domain (TMD) from KEX2 gene sequence. Constructed KEX2 is the KEX2-650 variant with TMD deletion and cytoplasmic domain. The recombinant plasmid was constructed through site-directed mutagenesis using FP-Kex2-699 and RP-Kex2-650 primers, including the BamHI site for plasmid religation. PCR site-directed mutagenesis produces an amplicon DNA with an expected length of 5551 bp. After restriction (BamHI) and religation, the plasmid was reintroduced into Escherichia coli DH5? and obtained 16 colonies. Verification PCR target gene showed that clones number 9 produced an amplicon of expected length (646 bp). DNA sequencing analysis confirmed that TMD was removed from the gene construct to form the KEX2-650 construct.