Pichia pastoris ile hücredışı rekombinant insan büyüme hormonu üretimi.

Pyrococcus furiosus extracellular α-amylase is a hyperthermostable glucosyl hydrolyzing enzyme which shows unique biochemical properties that may have impact on improving starch hydrolysis process; however, it is insignificantly expressed in its native archaeal host. In this study, it was aimed to express the P. furiosus extracellular alpha-amylase (PFA) in Pichia pastoris, which is a well-recognized overexpression host used in production of heterologous proteins. In this context, first, P. furiosus was grown under anaerobic conditions in capped bottles for t= 12 h at T=90°C and then its genomic DNA was isolated. PFA coding cDNA frame was amplified using two specifically designed oligonucleotides and cloned into pPICZαA expression vector. Then wild type P. pastoris X-33 cells were transfected with pPICZαA::PFA construct. In shake flask production medium, existence of recombinant PFA activity was tested and biochemical characterization of the recombinant product was done. This was the first time PFA is expressed in an eukaryotic host. Optimum working temperature and pH of the rPFA were found to be 95 °C and within the range of 4.5-6.5, respectively. rPFA is independent to metal ions and inhibition by production medium of P. pastoris was observed, in presence of divalent metal ions. Although Saccharomyces cerevisiae α-factor secretion signal was fused to the N terminal of rPFA, minute amount of extracellular secretion was detected but the majority of the enzymatic activity remained in the intracellular medium. The best producer strain was selected by measuring α-amylase activity in cell extracts by DNS method. Effects of pH on cell growth and recombinant protein production were determined by shake flask experiments and maximum of 4800 U/l rPFA was detected with 7.30 g/l wet cell density in pH=6 buffered medium. In order to achieve higher rPFA production, two bioreactor experiments were designed at two different pH operation conditions, namely pH=4 and pH=5, in a working volume of 1 L. The dissolved oxygen tension was kept over 20% and predetermined exponential methanol feeding strategy was employed in order to fix specific cell growth rate, µ, at 0.03 h-1. At pH=4 operation, maximum of 73,400 U/l α-amylase activity was detected at the t=27 h of production phase when the wet cell density was 209 g/l.M.S. - Master of Scienc

P.FURİOSUS ɑ-AMİLAZININ MODİFİYE BAKTERİYEL PROMOTER KULLANILARAK B.LİCHENİFORMİS İLE YÜKSEK MİKTARDA EKSPRESYONU

P.FURİOSUS ɑ-AMİLAZININ MODİFİYE BAKTERİYEL PROMOTER KULLANILARAK B.LİCHENİFORMİS İLE YÜKSEK MİKTARDA EKSPRESYON

FEN BİLİMLERİ ENSTİTÜSÜ/LİSANSÜSTÜ TEZ PROJESİ

P. PASTORİS İLE HİPERTERMOFİLİKK PROTEİN EKSPRESYONUNDA KARŞILAŞILAN SORUNLARIN BELİRLENMESİ VE REKOMBİNANT PYROCOCCUS FURİOUSUS ALFA - AMİLAZ ÜRETİM PROSESİSİNİN İYİLEŞTİRİLMES

FEN BİLİMLERİ ENSTİTÜSÜ/LİSANSÜSTÜ TEZ PROJESİ

P.PASTORİS İLE HİPERTERMOFİLİKK PROTEİN EKSPRESYONUNDA KARŞILAŞILAN SORUNLARIN BELİRLENMESİ VE REKOMBİNANT Pyrococcus Furiousus ALFA-AMİLAZ ÜRETİM PROSESİNİN İYİLEŞTİRİLMES

P. PASTORİS İLE HİPERTERMOFİLİKK PROTEİN EKSPRESYONUNDA KARŞILAŞILAN SORUNLARIN BELİRLENMESİ VE REKOMBİNANT PYROCOCCUS FURİOUSUS ALFA - AMİLAZ ÜRETİM PROSESİNİN İYİLEŞTİRİLMESİ

P. PASTORİS İLE HİPERTERMOFİLİKK PROTEİN EKSPRESYONUNDA KARŞILAŞILAN SORUNLARIN BELİRLENMESİ VE REKOMBİNANT PYROCOCCUS FURİOUSUS ALFA - AMİLAZ ÜRETİM PROSESİNİN İYİLEŞTİRİLMES

FEN BİLİMLERİ ENSTİTÜSÜ/LİSANSÜSTÜ TEZ PROJESİ

P. PASTORIS İLE HİPERTERMOFİLİKK PROTEİN EKSPRESYONUNDA KARŞILAŞILAN SORUNLARIN BELİRLENMESİ VE REKOMBİNANT PYROCOCCUS FURIOUSUS ALFA-AMİLAZ ÜRETİM PROSESİNİN İYİLEŞTİRİLMES

Codon optimization of xylA gene for recombinant glucose isomerase production in Pichia pastoris and fed-batch feeding strategies to fine-tune bioreactor performance

The objectives of this work are the optimization of the codons of xylA gene from Thermus thermophilus to enhance the production of recombinant glucose isomerase (rGI) in P. pastoris and to investigate the effects of feeding strategies on rGI production. Codons of xylA gene from T. thermophilus were optimized, ca. 30 % of the codons were replaced with those with higher frequencies according to the codon usage bias of P. pastoris, codon optimization resulted in a 2.4-fold higher rGI activity. To fine-tune bioreactor performance, fed-batch bioreactor feeding strategies were designed as continuous exponential methanol feeding with pre-calculated feeding rate based on the pre-determined specific growth rate, and fed-batch methanol-stat feeding. Six feeding strategies were designed, as follows: (S1) continuous exponential methanol- and pulse- sorbitol feeding; (S2) continuous exponential methanol- and peptone- feeding; (S3) continuous exponential methanol- and pulse- mannitol feeding; (S4) continuous exponential methanol- and peptone- feeding and pulse-mannitol feeding; (S5) methanol-stat feeding by keeping methanol concentration at 5 g L-1; and, (S6) methanol-stat feeding by keeping methanol concentration at 5 g L-1 and pulse-mannitol feeding. The highest cell and rGI activity was attained as 117 g L-1 at t = 66 h and 32530 U L-1 at t = 53 h, in strategy-S5. The use of the co-substrate mannitol does not increase the rGI activity in methanol-stat feeding, where 4.1-fold lower rGI activity was obtained in strategy-S6. The overall cell yield on total substrate was determined at t = 53 h as 0.21 g g(-1) in S5 strategy