Rekayasa protein actinidin dan ekspresinya pada khamir Saccharomyces cerevisiae

ASTUTI, Agung, Ir. Triwibowo Yuwono, PhD

2001 | Tesis | S2 Bioteknologi

Enzim protease banyak diperlukan di berbagai industri. Actinidin merupakan salah satu protease kuat dihasilkan dari buah Kiwi yang bersifat intraselular. Untuk produksi enzim protease actinidin perlu dilakukan rekayasa terhadap gen pengkode protein actinidin agar dapat diekspresi dan sekresi sehingga bersifat ekstraselular. Dalam penelitian ini dilakukan rekayasa gen actinidin dengan menghilangkan asam amino QR pada C-terminal extension melalui teknik PCR. Ekspresi dan sekresi varian DNA actinidin R4 dipelajari dengan melakukan kloning pada khamir Succhuromyces cerevisiue DBY 746. Varian DNA clctinidjn R4 hasil amplifikasi PCR disisipkan ke sisi restriksi Hindm-SuZI plasmid pYSW (vektor ekspresi-sekresi S. cerevisiue) sehingga dihasilkan plasmid rekombinan pYSV9-R4 kemudian ditransformasikan ke S. cerevisiue DBY 746. Ekpresi varian DNA actinidin R4 dianalisis dengan elektroforesis SDS-PAGE dan immunoblotting. Penelitian ini telah berhasil mendapatkan klon varian DNA actinidin R4 pada khamir S. cerevisiue DBY 746. Protein ekstraselular dengan BM sekitar 23.6 kD berhasil terekspresi pada khamir S. cerevisiue DBY 746 yang membawa plasmid rekombinan pYSV9-R4 dalam medium YEPD cair. Analisis immunoblotting terhadap protein supernatan tidak menunjukkan ada protein yang bereaksi spesifik dengan antibodi-anti-actinidin.

Actinidin is an intracellular cysteine protease found in the fruit of Chinese gooseberry (Actinidiu chinensis) or Kiwi fruit. A variant of actinidin-encoding DNA sequence has been constructed by amplifying a specific region of the full-length actinidin cDNA using PCR method. Amplification was targeted to construct an actinidin-encoding DNA sequence consisting of mature actinidin-encoding DNA plus part of the N-terminal extension (carrying the QRTN motif) and part of the C-terminal extension without QR sequence motif. The amplified fragment was then cloned in an expression-secretion vector (pYSV9) and expressed in Succhuromyces cerevisiae. The results of this study demonstrated that the cloned fragment had a smaller size as compared to the PCR amplification product, suggesting that a rearrangement event occured during the cloning process. Yeast culture supernatant was analysed for expression of actinidin by using anti-actinidin polyclonal antibody raised in chicken. The expression of the cloned fragment in S. cerevisiae, however, did not result in any detectable expression product on the immunoblot.

Kata Kunci : Bioteknologi,Rekayasa Protein Actinidin, Actinidin, expression and secretion, Saccharomyces cerevisiae