Production Optimation of Aspmt Insulin Recotnbinant Precursor in Pichia pastoris X-33

• Main Author: Mohammad Abdillah, Alfin
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/38586
• Institution: Institut Teknologi Bandung

Diabetes b a chronic metabolic disorder characterized b) high blood sugar le\els accompanied b) metabolic di sorders as a result of insufficiency of insulin function. One type of analog in sulin recombinant that is characterized by rapidl} acting ana logues is aspart insulin. The aim of thi s stud} \\US to construct Pichia pas/oris X -33 yeast CUlT) i ng a aspart insulin recombi nant precursor gene (Asp!) and optim i ze prod uction of aspart insulin recombinant precursors in P. pwlori\ X-33 using Re::.pon::.c Surface Mt:thodolog)' (RSM). P.pa\,lori. X-33 "a::, lran::.formed \\ ith plasmid pPICZaA-.-1.\p/ \\hich encodes a polpeptide consisting of 58 amino acid residues using the electroporat i on method. The P. pastoris X-33 transformant which carrying multi-copies of the Aspl gene "as selected on t he YPD grm\th medium (Yeast extract­ Peptone-Dex.trose) containing Zeoc in 500 )lg I mL. l .000 )lg I mL. 1.500 pg I mL. and :2.000 tg I rnL. There \vere 49 final colonies grO\vn from 49 selected colon i e . 638 pa i rs of base DN A fragments obtained from Pol)merized Chain Reaction (PCR) col onies "h ich shov.ed the size of' a::,part it ::> ulin rCOIIlbinant pn: ,;ur::,Lll' gene!>. ::;_5 ,;ok)J1ie!> have been l:Oil (i nned to c..:ontai11 t he ,b• pi gene \\ith the Mut gcnot)pc. Analysis ofSDS-PAGE e:\.pression from 3. 15. 34. 50. and 51 coloniC!.> shov.cd that coloni 34 had the opti mum recombinant in ulin aspa rt precursor band. The optimum incubation period of 3% (\ lv) methanol•induction in the production of a:.part insulin recombinant precursors in P. pas/oris X-33-Aspl-34 was 72 hours. The production of P. pas/oris X-33-Aspl-34 \vas optimized in the Buffered Methanol-complex Med i um (BMMY) using RSM with a Centra l Composite Design (CCD) four level. P. pastoris X-33- Aspl-34 colonies were incubated using a sha!...ing flask for 72 hours. The parameters used to optimize the production conditione; are. the concentration of methanol induction. inilial oo( •­ :omparison of aeration. and pH of the gro\\th medium. Aspart insulin recombi nant precursor expression \vas analyzed using SDS-PAGE and quantified using a densitometer to determine t he aspart in::, ulin precur::,or :onct:nlration produ :ed. The aspart insulin pre :ursor concentrat ion that obta ined \\as used as a response for RSM and ana l) Led using M initab 18 software. Based on the re ults oft he optimi zati on anal) sis using RSM.1he optimu m prod uction conditi ons for the C:\.pre sion of aspart i11::>ulin recombinant precur:.ors in P. pastoris X-33 are pH 6.3. 3°o metha1wl concentration. inilial 00•.()0 50. and aeration ratio of 8 m L I 00 mL.