Karakterisasi Protein Disulfida Isomerase Hasil Pemurnian Sebagian Dari Saccharomyces Cerevisiae [pUKC470]

Protein disulfida isomerase (PDI) merupakan multi enzim yang berfungsi mengkatalisis reaksi redoks dan reaksi isomerisasi ikatan disulfida dalam berbagai protein sekresi. PDI telah diisolasi dari Sacharomyces cerevisiae dengan aklivitas spesifik kecil. Sehubungan dengan over-ekspresi gen PD/I, telah dikonstruksi plasmid pUKC470 yang mengandung gen PD/I S. cerevisiae, tetapi sifat-sifat katalitik PDl S. cerevisiae hasil over ekspresi tersebut belum diketahui. Pada penelitian ini dilakukan karakterisasi enzim PDI hasil pemurnian parsial dari S. cerevisiae (pUKC470] untuk memperoleh informasi kinetika enzim tersebut, khususnya terhadap substrat insulin. Enzim PDI dimurnikan dari cell free extract transforman menggunakan metode fraksinasi amonium sulfat 60-80% diikuti dengan kromatografi kolom penukar anion DEAE-Sephacel. Kondisi optimum enzim protein disulfide isomerase PDI hasil pemurnian sebagian dari S. cerevisiae [pUKC470] tersebut berturut-turut adalah waktu 13 menit, pH 7,5 dan suhu 37'C. Data kinetik menunjukkan bahwa PDI termasuk enzim alosterik dengan nilai Vmaks enzim sekitar 98unit/ml dan Km apparent terhadap substrat insulin 8,0x10-2 mM. Bacitracin merupakan modulator negatif PDI

Isolation And Partially Purification Of Protein Disulfida Isomerase From Saccharomyces Cerevisiae [pUKC470]

Protein disulphide isomerase (PDI) is a multi-enzyme involved in catalyzing redox and isomerization reactions of clisulphide bonds in secretory proteins. This investigation is focused on the isolation and partially purification of PDI from Saccharomyces cerevisiae [p UKC4 70] in order to elucidate mechanism of action of PDI by characterizing kinetics of the enzyme. The specific activity of PDI isolated from transformant increased by 17 fold compared to the wild type Saccharomyces cerevisiae W303. The purification of PDI from transformant using ammonium sulphate fractionation followed by ion exchange chromatography on DEAE-Sephacel revealed that the specific activity of PDI increased to 255.28 unit per mg, a degree of purity 137 fold compared to the cells free extract, and yield of 41 %

The b’x Region of Yeast Protein Disulfide Isomerase is Not Essential for Saccharomyces cerevisiae Viability at 30 °C

Protein disulfide isomerase (PDI) catalyzes thiol oxidation, reduction and isomerization of disulphide bond of cell surface and secreted proteins. Yeast PDI1 consists of two catalytic domains (a and a’) which are separated by two non-catalytic domains (b and b’), and a x region linked the b’ and a domains. The b’ domain is important for the non-covalent binding of partially folded protein. To understand the contribution of b’ domain and x-linker of yeast PDI1 we have deleted the b’x and investigated its functional role in vitro and in vivo. Yeast PDI1 without b’x region retained only 50% activity and became more sensitive toward Proteinase K. Interestingly, yeasts containing full length PDI1 and pdi1Db’x showed approximately the same growth rate. However, the yeast pdi1Db’x mutant growth impaired severely at 37 °C compared to that of the full length PDI1. Our results suggested that the a-b-a’-c domains of PDI seems to be sufficient to support the growth of yeast cells in normal condition, but the b’x region might be essential in assisting refolding of highly accumulated unfolded protein at high temperature (37 °C)