Dissertation > Excellent graduate degree dissertation topics show

Influence of Culture Conditions on the Secretory Expression of Porcine Insulin Precursor and Physiological Characterization of Multi-copy P.pastoris

Author: Wang
Tutor: ZhuangYingPing；GuoMeiJin
School: East China University of Science and Technology
Course:
Keywords: Pichia pastoris gene copy number transcriptional analysis porcine insulin precursor chaperone protein disulfide isomerase culture condition
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 69
Quote: 0
Read: Download Dissertation

Abstract

Pichia pastoris is a well-established yeast host in both basic research and industrial protein production for the production of heterologous proteins. However, studies on physiologic characterization of Pichia pastoris cells under certain bioprocess-relevant conditions are still limited. In this thesis, there is a major concern to better understand the influence of environmental factors on the foreign protein productivity of the methylotrophic yeast Pichia pastoris. Taken porcine insulin precursor (PIP) as a foreign protein model, the impact of cultivation temperature, dissolved oxygen (DO) and over-expression of chaperone PDI (Protein Disulfide Isomerase) on the physiology and productivity of PIP protein secreted by P. pastoris strains with different foreign gene dosage was investigated in shake flask culture and/or in fed-batch culture in a 50L fermentor. The results were as follows:1) The PIP production mulit-copy P. pastoirs strains A2 with 12 PIP gene copies and A3 with 18 PIP gene copies were cultivated at 25℃and 30℃in shake flask and in a 50L fermentor, respectively. Analysis of transcriptional levels of key genes involvs in physiologies of cells and PIP production was also performed.The results showed that at lower tempreture(25℃), PIP production of A2 and A3 was increased by 40%and 32%, respectively, and the viability of recombinant strains A3 cells was also increased by 20%compared with these cultivated at 30℃. In the case of A3 strain cultured at 25℃, transcriptional levels of gene KAR2 (encoding binding protein) and PDI1 (encoding protein disulfide isomerase) were reduced by 35%and 27%, GLR(encoding glutathione reductase) and TRR1 (encoding thioredoxin reductase)were reduced by 16%as well, which involves in oxidative stress response and molecular chaperones. The findings indicate that protein folding stress and oxidative stress response in the high-copy strain cells are generally decreased at lower cultivation temperatures, possibly, led to about 40%increases in heterologous protein production by multi-copy P. pastoris host cells.(2) The PIP production mulit-copy P. pastoirs strains were cultivated in 50ml,100ml and 200ml medium volume each in a 500ml shake flask, respectively.With the medium volume increasing, the values of PIP/OD600(mg/L) of both A2 and A3 decreased by 36%and 28%, respectively. Meanwhile, it was observed that the lowest values both of PIP/OD/copy number (mg/L/copy) and of methanol consumption amount were achieved by A3 strain. Transcriptional levels of gene GAP (encoding glyceraldehyde-3-phosphate dehydrogenase in glycolysis) was reduced by 16%,CIT1 (encoding citrate synthase, a key enzyme in tricarboxylic acid cycle) was reduced by 30%, ZWF1 (encoding glucose-6-phosphate dehydrogenase, the first key enzyme in pentose phosphate pathway) was reduced by 15%, that involves in yeast methanol central metabolic pathways. It reveals that the metabolic flux through the TCA-cycle was reduced by reduction of dissolved oxygen (DO) supply, resulting in a decline of heterologous PIP protein secretion by about 28%, especially, in high-PIP-copy P. Pastoris host cells.(3) To explore effect of PDI over-expression on foreign protein production in high-copy strain cells, a strain A2p harboring PDI expression vector was constructed based on strain A2. The results showed that over-expression of chaperone PDI failed to improve the PIP expression, resulting from the occurrence of more severe oxidative stress in cells due to 60%higher in ROS level generated by PIP protein folding (the formation of protein disulfide bonds),25%lower cell viability and 38%reduced PIP production in comparison with those in control A2 cells.

Related Dissertations

CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
© 2012 www.DissertationTopic.Net Mobile