Amino acid content of recombinant proteins influences the metabolic burden response
Amino acid content of recombinant proteins influences the metabolic burden response
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DOI：
10.1002/bit.20436
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发表时间：
2005-04-05
影响因子：
3.8
通讯作者：
Gill, RT
中科院分区：
文献类型：
--
作者：
Bonomo, J;Gill, RT
在大肠杆菌中生产重组蛋白通常会导致戏剧性的细胞应激反应，最好的特征是总体细胞适合性下降。我们确定，重组蛋白的初级序列(氨基酸序列)本身在缓解这种反应方面起着重要作用。为此，我们以39-40氨基酸防御素类蛋白质为模型，创建了两个多肽，它们只包含大肠杆菌中最少的五个氨基酸(PepAA；His，Trp，Tyr，Phe，Met)或大多数(Pepco：Ala，Glu，Gln，Asp，Asn)丰富的氨基酸。我们发现，与Pepco、我们的模型防御素蛋白MGD-1或pET-3D载体骨架中包含的26个氨基酸的多肽相比，PepAA的过表达导致生长速度急剧下降。我们使用Affymetrix E.coli基因芯片进一步确定，这些模型系统的全基因组转录反应之间的差异最好地表现为其产物参与翻译、运输或代谢功能的基因的表达发生变化，而不是应激反应基因。基于这些结果，我们证实，与评估的其他模型多肽相比，过度表达PepAA的细胞的翻译效率显著降低。(C)2005年威利期刊公司。
Recombinant protein production in Escherichia coli often results in a dramatic cellular stress response best characterized by a decrease in overall cell fitness. We determined that the primary sequence (the amino acid sequence) of the recombinant protein alone plays an important role in mitigating this response. To do so, we created two polypeptides, modeled after the 39-40 amino acid Defensin class of proteins, which contained exclusively the five least (PepAA; His, Trp, Tyr, Phe, Met), or most (PepCO: Ala, Glu, Gln, Asp, Asn) abundant amino acids in E. coli. We determined that overexpression of PepAA resulted in a drastic decrease in growth rate compared to overexpression of PepCO, our model Defensin protein MGD-1, or the 26 amino acid polypeptide contained within the pET-3d vector backbone. We further determined, using Affymetrix E. coli gene chips, that differences among the whole-genome transcriptional responses of these model systems were best characterized by altered expression of genes whose products are involved in translation, transport, or metabolic functions as opposed to stress response genes. Based on these results, we confirmed that translation efficiency was significantly reduced in cells overexpressing PepAA compared with the other model polypeptides evaluated. (c) 2005 Wiley Periodicals, Inc.