Yantsevich A V, Dzichenka Ya V, Ivanchik A V, Shapiro M A, Trawkina M, Shkel T V, Gilep A A, Sergeev G V, Usanov S A
Prikl Biokhim Mikrobiol. 2017 Mar-Apr;53(2):173-87. doi: 10.1134/s000368381702017x.
Contaminating proteins have been identified by “shotgun” proteomic analysis in 14 recombinant preparations of human membrane heme- and flavoproteins expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Immobilized metal ion affinity chromatography of ten proteins was performed on Ni2+-NTA-sepharose 6B, and the remaining four proteins were purified by ligand affinity chromatography on 2',5'-ADP-sepharose 4B. Proteomic analysis allowed to detect 50 protein impurities from E. coli. The most common contaminant was Elongation factor Tu2. It is characterized by a large dipole moment and a cluster arrangement of acidic amino acid residues that mediate the specific interaction with the sorbent. Peptidyl prolyl-cis-trans isomerase SlyD, glutamine-fructose-6-phosphate aminotransferase, and catalase HPII that contained repeating HxH, QxQ, and RxR fragments capable of specific interaction with the sorbent were identified among the protein contaminants as well. GroL/GroS chaperonins were probably copurified due to the formation of complexes with the target proteins. The Ni2+ cations leakage from the sorbent during lead to formation of free carboxyl groups that is the reason of cation exchanger properties of the sorbent. This was the putative reason for the copurification of basic proteins, such as the ribosomal proteins of E. coli and the widely occurring uncharacterized protein YqjD. The results of the analysis revealed variation in the contaminant composition related to the type of protein expressed. This is probably related to the reaction of E. coli cell proteome to the expression of a foreign protein. We concluded that the nature of the protein contaminants in a preparation of a recombinant protein purified by immobilized metal ion affinity chromatography on a certain sorbent could be predicted if information on the host cell proteome were available.
通过“鸟枪法”蛋白质组学分析，在14种在大肠杆菌中表达并通过固定化金属离子亲和色谱法纯化的重组人膜血红素蛋白和黄素蛋白制剂中鉴定出了污染蛋白。十种蛋白质在Ni2 + -NTA-琼脂糖6B上进行固定化金属离子亲和色谱，其余四种蛋白质通过在2'，5'-ADP-琼脂糖4B上的配体亲和色谱法纯化。蛋白质组学分析检测到来自大肠杆菌的50种蛋白质杂质。最常见的污染物是延伸因子Tu2。它的特征在于大的偶极矩和酸性氨基酸残基的簇状排列，这些残基介导与吸附剂的特异性相互作用。在蛋白质污染物中还鉴定出了含有能够与吸附剂特异性相互作用的重复HxH、QxQ和RxR片段的肽基脯氨酰顺反异构酶SlyD、谷氨酰胺-果糖-6-磷酸转氨酶和过氧化氢酶HPII。GroL / GroS伴侣蛋白可能由于与靶蛋白形成复合物而被共纯化。吸附剂在过程中Ni2 +阳离子泄漏导致游离羧基的形成，这是吸附剂具有阳离子交换特性的原因。这是共纯化碱性蛋白质（如大肠杆菌的核糖体蛋白和广泛存在的未表征蛋白YqjD）的推测原因。分析结果显示，污染物组成因所表达蛋白质的类型而异。这可能与大肠杆菌细胞蛋白质组对外源蛋白表达的反应有关。我们得出结论，如果有宿主细胞蛋白质组的信息，那么通过在特定吸附剂上进行固定化金属离子亲和色谱法纯化的重组蛋白制剂中蛋白质污染物的性质是可以预测的。
