Proteomic analysis of contaminants in recombinant membrane hemeproteins expressed in E. coli and isolated by metal affinity chromatography
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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.
Keywordshemeprotein recombinant protein chaperonin proteomics mass spectrometry immobilized metal ion affinity chromatography
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- 15.Chudaev, M.V. and Usanov, S.A., Biochemistry (Moscow), 1997, vol. 62, no. 4, pp. 401–411.Google Scholar
- 18.Shkel’, T.V., Vasilevskaya, A.V., Gilep, A.A., Chernovetskii, M.A., Luk’yanenko, I.G., and Usanov, S.A., Trudy BGU, 2013, vol. 8, no. 1, pp. 152–158.Google Scholar