A systematic analysis of the expression of the anti-HIV VRC01 antibody in Pichia pastoris through signal peptide optimization.
A systematic analysis of the expression of the anti-HIV VRC01 antibody in Pichia pastoris through signal peptide optimization.
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DOI：
10.1016/j.pep.2018.03.013
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发表时间：
2018-09
影响因子：
1.6
通讯作者：
Polizzi KM
中科院分区：
文献类型：
--
作者：
Aw R;McKay PF;Shattock RJ;Polizzi KM
巴斯德毕赤酵母（Pichia pastoris）（Komagataella phaffi）已经用于重组蛋白生产超过30年，迄今为止报道了超过5000种蛋白。然而，抗体的产量通常较低。我们已经评估了分泌信号肽对产生广泛中和抗体（VRC 01）以增加产量的影响。组合评价了11种不同的信号肽（包括鼠IgG 1信号肽）对抗体滴度的影响。鉴定了使用不同信号肽组合的菌株，其分泌的VRC 01水平比先前最佳分泌子高约2-7倍，在摇瓶表达中的最高产量为6.50 mg L-1。有趣的是，确定了当鼠IgG 1信号肽与轻链融合时达到最高产率，当与重链融合时，几种不同的信号肽导致高产率。最后，我们已经评估了使用2A信号肽来创建双顺反子载体以试图减少负担并增加转化效率的效果，但发现与使用两个独立载体相比，其产量降低。在巴斯德毕赤酵母中VRC 01抗体的产量增加了2-7倍。在轻链上使用鼠IgG 1信号肽导致更高的产率。双顺反子载体的使用没有增加VRC 01的产量。
Pichia pastoris (Komagataella phaffi) has been used for recombinant protein production for over 30 years with over 5000 proteins reported to date. However, yields of antibody are generally low. We have evaluated the effect of secretion signal peptides on the production of a broadly neutralizing antibody (VRC01) to increase yield. Eleven different signal peptides, including the murine IgG1 signal peptide, were combinatorially evaluated for their effect on antibody titer. Strains using different combinations of signal peptides were identified that secreted approximately 2–7 fold higher levels of VRC01 than the previous best secretor, with the highest yield of 6.50 mg L−1 in shake flask expression. Interestingly it was determined that the highest yields were achieved when the murine IgG1 signal peptide was fused to the light chain, with several different signal peptides leading to high yield when fused to the heavy chain. Finally, we have evaluated the effect of using a 2A signal peptide to create a bicistronic vector in the attempt to reduce burden and increase transformation efficiency, but found it to give reduced yields compared to using two independent vectors. Production of VRC01 antibody in Pichia pastoris was increased 2–7 fold. Using the murine IgG1 signal peptide on the light chain results in higher yields. The use of bicistronic vectors did not increase the yield of VRC01.
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影响因子：
6.4
作者：
Aw R;Polizzi KM
通讯作者：
Polizzi KM
DOI：
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发表时间：
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影响因子：
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