INCORPORATION OF NORLEUCINE AT METHIONINE POSITIONS IN RECOMBINANT HUMAN MACROPHAGE-COLONY-STIMULATING FACTOR (M-CSF, 4-153) EXPRESSED IN ESCHERICHIA-COLI - STRUCTURAL-ANALYSIS
INCORPORATION OF NORLEUCINE AT METHIONINE POSITIONS IN RECOMBINANT HUMAN MACROPHAGE-COLONY-STIMULATING FACTOR (M-CSF, 4-153) EXPRESSED IN ESCHERICHIA-COLI - STRUCTURAL-ANALYSIS
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DOI：
10.1021/bi00180a032
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发表时间：
1994-04-12
期刊：
影响因子：
2.9
通讯作者：
SHACKLETON, CHL
中科院分区：
文献类型：
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作者：
RANDHAWA, ZI;WITKOWSKA, HE;SHACKLETON, CHL
重组巨噬细胞集落刺激因子(rm-CSF，4-153)的17.5 kDa截短型在大肠杆菌中的表达因去甲亮氨酸取代蛋氨酸残基而变得复杂。为了检测和定量这一错误翻译事件，用氨基酸分析、自动Edman氨基酸测序和电喷雾质谱仪分析了完整蛋白质和S-羧胺甲基化蛋白质。此外，还对内切酶Glu-C产生的多肽进行了氨基酸序列测定、高效液相色谱和电喷雾电离质谱分析。不同批次的RM-CSF中去甲亮氨酸的取代度在0%~20%之间。在计算去甲亮氨酸在蛋氨酸位置上的取代程度时，需要考虑蛋氨酸残基的相对不稳定性。完整的RM-CSF的质谱学可以检查多取代蛋氨酸在去甲亮氨酸物种中的分布，并使去甲亮氨酸掺入的检测和定量降至约3%的水平。反相高效液相色谱/电喷雾电离电离质谱仪获得的目标分子离子的选择性离子色谱图为去甲亮氨酸多肽的检测和定量提供了一种可靠、快速的方法。去甲亮氨酸残基均匀分布在所有四个蛋氨酸位置(10、27、61和65)。用其结构去亮氨酸类似物取代蛋氨酸对复性的RM-CSF二聚体的活性没有任何影响。
Expression of the 17.5-kDa truncated form of human recombinant macrophage colony stimulating factor (rM-CSF, 4-153) in Escherichia coli is complicated by the replacement of methionine residues by norleucine. In order to detect and quantitate this mistranslational event, the intact and the S-carboxyamidomethylated proteins were analyzed by amino acid analysis, automated Edman amino acid sequencing, and electrospray mass spectrometry. In addition, the endoproteinase Glu-C generated peptides were subjected to amino acid sequencing, high-performance liquid chromatography, and electrospray ionization mass spectrometry. The extent of norleucine substitution in different batches of rM-CSF varied between 0% and 20%. The relative instability of methionine residues needs to be considered when calculating the extent of norleucine substitution at methionine positions. The mass spectrometry of the intact rM-CSF allowed for examination of the distribution of multiply substituted methionine to norleucine species, and it enabled detection and quantitation of the norleucine incorporation down to the approximately 3% level. Selective ion chromatograms of molecular ions of interest obtained in reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry of proteolytic fragments offered a reliable and fast method of detection and quantitation of norleucine-containing peptides. Norleucine residues were uniformly distributed among all four methionine positions (10, 27, 61, and 65). A substitution of methionine by its structural norleucine analog does not have any effect on the activity of the refolded rM-CSF dimers.