SUBKLONING GEN -L-ARABINOFURANOSIDASE (abfA) DALAM VEKTOR EKSPRESI pYES2

Abstract

A Gene encoding -L-arabinofuranosidase (abfa) that had been cloned into recombinant E. coli
DH5a/pTP510 was subcloned into Saccharomyces cerevisiae yeast system. The aim of subcloning of abfA gene into
S. cerevisiae is to express the thermostable -L-arabinofuranosidase (AbfA) enzyme in the host that is often termed
as Generally Recognized As Safe (GRAS), so that this enzyme earn the broader application like in food and beverage
industries. The gene of abfA was subcloned by the amplification of Polymerase Chain Reaction (PCR) from plasmid
pTP510 templat. A pair of primers, pFSacI-Af (forward) and pXhoI-Af (reverse) from designed this research was
used for the amplifcation. The PCR condition was performed as follows : beginning denaturation at 94oC for 5 min;
PCR cycle 30 times that consisted of denaturation (94oC for 1 min), annealing (55oC for 30 s), and
polymerization/extension at 72oC for 2 min; and than final extension at 72oC for 7 min. The abfA gene, resulted was
inserted between GAL1 promoter and CYT1 terminator in the pYES2 expression vector. This ligation product was
transformed into E. coli TOP10 host. Restriction analysis of recombinant plasmid from this construction, the
designated as plasmid pY-Af, showed the expected size, about 7,4 kb, which was the summation of sizes of parental
plasmid ( 5,9 kb) and fragment of DNA insert (1,5 kb).