Produksi Actinidin rekombinan yang diekspresikan pada Khamir Saccharomyces cerevisiae

ANGGRENI, Anak Agung Made Dewi, Dr.Ir. Triwibowo Yuwono

2005 | Tesis | S2 Bioteknologi

Actinidin merupakan protease sistein yang dihas ilkan dari buah kiwi (Actinidia chinensis). Pada penelitian sebelumnya, beberapa gen yang mengkode actinidin telah diklon pada Saccharomyces cerevisiae untuk mengontrol ekspresi gen dan mengarahkan protein ke medium ekstraselular. Selanjutnya telah juga didapatkan bahwa hasil ekspresi dan sekresi actinidin rekombinan pada S. cerevisiae BF307-10 yang membawa plasmid rekombinan (pYSV9~R1) menggunakan volume 500 ml sangat sedikit. Tujuan penelitian ini adalah memperbesar jumlah actinidin rekombinan yang dihasilkan. Transforman di dapat dari penelitian sebelumnya. Ekspresi protein actinidin rekombinan dilakukan dengan menumbuhkan transforman (S. cerevisiae strain BF307-10 yang membawa plasmid rekombinan pYSV9~R1) pada medium YEPD volume 5 x 500 ml. Protein supernatan dipanen pada fase eksponensial dan fase stasioner. Supernatan protein difraksionasi dengan pengendapan secara bertingkat dengan ammonium sulfat tingkat kejenuhan 30, 50, 70, dan 90 %. Hasil fraksionasi kemudian didialisis dan dikeringbekukan. Hasil penelitian menunjukkan bahwa volume medium 5 x 500 ml menghasilkan jumlah protein yang lebih besar. Produksi actinidin rekombinan diperoleh pada fase pertumbuhan eksponensial sedangkan pada fase pertumbuhan stasioner actinidin rekombinan tidak terdete ksi. Hasil immunobloting menunjukkan bahwa actinidin rekombinan terdeteksi pada sampel yang berasal dari transforman pYSV9 ~R1 yang dipresipitasi dengan ammonium sulfat dengan tingkat kejenuhan 30 %. Protein tersebut mempunyai ukuran 27,4 kD. Fraksi protein yang mengandung actinidin rekombinan mempunyai aktivitas enzim protease. Dari hasil penelitian ini dapat disimpulkan bahwa produksi actinidin rekombinan berhubungan dengan fase pertumbuhan. Jumlah actinidin rekombinan yang dihasilkan meningkat dengan penambahan volume medium. Actinidin rekombinan terdeteksi pada medium yang dipanen pada fase pertumbuhan eksponensial.

Actinidin is a cysteine protease found in kiwi fruit (Actinidia chinensis). In previous study, variants of actinidin – encoding gene has been cloned in yeast Saccharomyces cerevisiae to drive the expression of the gene and direct the protein to extracellular space. It had also been indicated that the results of expression and secretion of recombinant actinidin in S. cerevisiae strain BF 307 – 10 carrying recombinant plasmid (pYSV9~R1) using 500 ml volume was very low. Therefore, the objective of this study was to increase the amount of recombinant actinidin produced in Saccharomyces cerevisiae . Transformant was obtained from previous study. The expression of recombinant actinidin protein was done by cultivating transformant (S. cerevisiae strain BF 307 – 10 carrying recombinant plasmid pYSV9~R1) in 5 x 500 ml of YEPD medium. Supernatant protein was harvested at the exponential and stationary growth phase. Supernatant protein was fractionated by successive precipitation level at saturation of 30, 50, 70, and 90 % of ammonium sulfate. The results of fractionation were then dialysed and freeze -dried. The results showed that 5 x 500 ml volume of medium produced larger amount of protein. Production of recombinant actinidin was acquired at exponential growth phase. However, at stationery growth phase, no recombinant actinidin was detected. Immunoblotting analysis demonstrated that recombinant actinidin was detected in the sample of pYSV9~R1 transformant precipitated by ammonium sulfate at 30 % saturation. The size of actinidin detected was 27,4 kD. Protein fraction containing recombinant actinidin also showed a protease activity. The result of this study thus demonstrated that produc tion of recombinant actinidin was growth phase – correlated. The amount of recombinant actinidin was also found increased with the increase of volume of medium. Recombinant actinidin was detected in the medium harvested at the exponential growth phase.

Kata Kunci : actinidin rekombinan, ekspresi, sekresi, Saccharomyces cerevisiae, aktu inkubasi, immunobloting, recombinant actinidin, expression, secretion, incubation time