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Study on Environmental and Control Conditions for Effective Fusion Protein Expression by Pichia Pastoris

Author: DuYiPing
Tutor: ShiZhongPing
School: Jiangnan University
Course: Biochemical Engineering
Keywords: Pichia pastoris Fusion protein Dissolved oxygen Methanol concentration
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 179
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Abstract

In this thesis, as a means to DO-Stat method and artificial neural network dynamic pattern recognition control (Artificial Neural Network Pattern Recognition based control, ANNPR-Ctrl), Pichia pastoris fed-batch culture production process of HSA-IL-2 fusion protein glycerol fed-batch control strategies and performance indicators carried out a comparative study. Particularly effective in cell growth phase-based online measurement method ANNPR-Ctrl DO (dissolved oxygen) / pH. It can be effective to increase the intensity of cell production, and to shorten the incubation time, so that the initiation of induction with methanol as the fusion protein of the inducing agent substantial advance. However, in the induction period, the methanol concentration must be controlled at a certain level in order to achieve efficient expression of the fusion protein. At the same time, recombinant Pichia pastoris (KM71, MutS) using methanol capacity is weak, unable to take advantage of the DO and pH to determine the excess or lack of status methanol. Therefore, the wire electrode of the induction period is the use of methanol to control the methanol stream plus the methanol concentration control within the setting range, to achieve efficient expression of the fusion protein. Using recombinant Pichia pastoris (KM71, MutS) expression of interleukin - 2 (HSA-IL-2) production of human albumin modified process, DO and methanol concentration is the key factor affecting the expression of the target protein. It was found that HSA-IL-2-producing bacteria DO demand is not very high, by using the air and combined adjusting stirring speed will be able to meet their demand of DO. 3 days after induction, in normal the DO (DO 20-60% of the lower concentration of methanol, the methanol concentration of 5-8G / L), and HSA-IL-2 expression in an amount of 23 mg / L, and 2.5 times for the expression amount of the shake flask; high DO, low methanol concentration (DO 80% -120%, methanol concentration of 2-5g / L), HSA-IL-2 expression levels may be further increased by 18% to reach 27mg / L; DO oscillation, low methanol concentration ( methanol concentration of 2-7g / L) conditions are not conducive to the expression of HSA-IL-2; under normal DO, a higher methanol concentration (DO 20-60%, methanol concentration of 15-23g / L), HSA-IL-2 expression The highest reached 44.6mg / L. Induced early high methanol concentration easily lead to bacterial poisoning is not conducive to the expression of the HSA-IL-2. In addition, in the induction phase, by restriction adding glycerol can effectively improve the breathing activity of the microbial cells, to promote cell growth, and alleviate the toxicity of methanol on cell. The expression of recombinant Pichia pastoris (GS115, Mut) fusion protein HSA-CP conducted a preliminary study. It was found that the high demand of the bacteria on DO. Pass oxygen-enriched air (50%, V / V) when the highest cell concentration of OD600 reached 600, 1.8 times the corresponding values ??of about the through-air. Compared with the HSA-IL-2-producing bacteria, the induction characteristics of the bacteria are also different. It utilizes methanol ability, a short induction time, in 5L fermentor inducing 15h, the amount of protein expression of HSA-CPs can reach the maximum value, and the shake flask level corresponding the largest amount of protein expression in the induction of 3 days to reach. The air is used when the highest expression level of HSA-CP was 108mg / L, slightly lower than the shake flask; enriched air with the highest expression level of HSA-CP can be improved doubled, to reach 237 mg / L.

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