Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes
Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes
复制标题
DOI：
10.1016/s0168-1656(03)00003-8
复制
发表时间：
2003-04-10
影响因子：
4.1
通讯作者：
Enfors, SO
中科院分区：
文献类型：
--
作者：
Jahic, M;Gustavsson, M;Enfors, SO
利用巴斯德毕赤酵母在高密度生物反应器中培养产生的融合蛋白(CBM)，在高密度生物反应器培养条件下，获得了由帕氏新天牛纤维素酶6A和南极假丝酵母脂肪酶B组成的融合蛋白。通过从甘油生长转到甲醇生长诱导产生该菌株。培养上清液中的脂肪酶活性几乎以恒定的速度增加，直到相当于1.3g L(-1)的CBM-Calb的值。然而，根据Western印迹分析，只有大约40%的产物是全长的。这种损失是由于CBM和Calb部分之间连接物中的蛋白质被切割所致。裂解由培养上清液中的丝氨酸蛋白酶催化。钙质部分通过细胞相关的蛋白分解而进一步缓慢降解。不同的策略被用来减少蛋白质的降解。以前缩短连接区的努力导致了稳定的蛋白质，但在生物反应器培养中的产物浓度降低了十倍(Gustavsson等人)。2001年，蛋白质工程。14、711-715)。添加富含酶底物竞争的培养基对CBM-Calb的蛋白分解没有影响。无论有无细胞存在，蛋白质分解反应的动力学都受pH的影响。当pH值低于5.0时，最快的反应，即连接子中的切割大大减少。将生物反应器培养物的pH值从5.0降低到4.0，全长产物的比例从40%增加到90%。通过将甲醇进料阶段的温度从30摄氏度降至22摄氏度，实现了进一步的改进。在最适pH和低温条件下，发酵上清液中可获得几乎全部的全长蛋白(1.5g L(-1))，且纯度较原培养物有较大提高。(C)2003 Elsevier Science B.V.保留所有权利。
A fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced by Pichia pastoris Mut(+) in high-cell density bioreactor cultures. The production was induced by switching from growth on glycerol to growth on methanol. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 1.3 g l(-1) of CBM-CALB. However, only about 40% of the product was of full-length according to Western blot analysis. This loss was due to a cleavage of the protein in the linker between the CBM and the CALB moieties. The cleavage was catalyzed by serine proteases in the culture supernatant. The CALB-moiety was subjected to further slow degradation by cell-associated proteolysis. Different strategies were used to reduce the proteolysis. Previous efforts to shorten the linker region resulted in a stable protein but with ten times reduced product concentration in bioreactor cultures (Gustavsson et al. 2001, Protein Eng. 14, 711-715). Addition of rich medium for protease substrate competition had no effect on the proteolysis of CBM-CALB. The kinetics for the proteolytic reactions, with and without presence of cells were shown to be influenced by pH. The fastest reaction, cleavage in the linker, was substantially reduced at pH values below 5.0. Decreasing the pH from 5.0 to 4.0 in bioreactor cultures resulted in an increase of the fraction of full-length product from 40 to 90%. Further improvement was achieved by decreasing the temperature from 30 to 22 degreesC during the methanol feed phase. By combining the optimal pH and the low temperature almost all product (1.5 g l(-1)) was obtained as full-length protein with a considerably higher purity in the culture supernatant compared with the original cultivation. (C) 2003 Elsevier Science B.V. All rights reserved.