Yefremova Yelena, Al-Majdoub Mahmoud, Opuni Kwabena F M, Koy Cornelia, Cui Weidong, Yan Yuetian, Gross Michael L, Glocker Michael O
Proteome Center Rostock, University Rostock Medical Center, Rostock, Germany.
J Am Soc Mass Spectrom. 2015 Mar;26(3):482-92. doi: 10.1007/s13361-014-1053-2. Epub 2015 Jan 6.
Mass spectrometric de-novo sequencing was applied to review the amino acid sequence of a commercially available recombinant protein G´ with great scientific and economic importance. Substantial deviations to the published amino acid sequence (Uniprot Q54181) were found by the presence of 46 additional amino acids at the N-terminus, including a so-called "His-tag" as well as an N-terminal partial α-N-gluconoylation and α-N-phosphogluconoylation, respectively. The unexpected amino acid sequence of the commercial protein G' comprised 241 amino acids and resulted in a molecular mass of 25,998.9 ± 0.2 Da for the unmodified protein. Due to the higher mass that is caused by its extended amino acid sequence compared with the original protein G' (185 amino acids), we named this protein "protein G'e." By means of mass spectrometric peptide mapping, the suggested amino acid sequence, as well as the N-terminal partial α-N-gluconoylations, was confirmed with 100% sequence coverage. After the protein G'e sequence was determined, we were able to determine the expression vector pET-28b from Novagen with the Xho I restriction enzyme cleavage site as the best option that was used for cloning and expressing the recombinant protein G'e in E. coli. A dissociation constant (K(d)) value of 9.4 nM for protein G'e was determined thermophoretically, showing that the N-terminal flanking sequence extension did not cause significant changes in the binding affinity to immunoglobulins.
采用质谱从头测序法对一种具有重要科学和经济意义的市售重组蛋白G´的氨基酸序列进行了分析。结果发现，该蛋白的氨基酸序列与已发表的氨基酸序列（Uniprot Q54181）存在显著差异，其N端额外存在46个氨基酸，包括一个所谓的“His标签”，以及分别在N端的部分α-N-葡糖酰化和α-N-磷酸葡糖酰化修饰。市售蛋白G´意外的氨基酸序列由241个氨基酸组成，未修饰蛋白的分子量为25,998.9±0.2 Da。由于其氨基酸序列比原始蛋白G´（185个氨基酸）更长，导致分子量更大，我们将这种蛋白命名为“蛋白G´e”。通过质谱肽图分析，所推测的氨基酸序列以及N端部分α-N-葡糖酰化修饰均得到了100%的序列覆盖确认。在确定了蛋白G´e的序列后，我们能够确定诺维信公司的表达载体pET-28b，其Xho I限制性酶切位点是用于在大肠杆菌中克隆和表达重组蛋白G´e的最佳选择。通过热泳法测定了蛋白G´e的解离常数（K(d)）值为9.4 nM，表明N端侧翼序列的延伸并未导致其与免疫球蛋白结合亲和力发生显著变化。
