基因工程产品的N末端均一性及其中残留的痕量杂质蛋白的分析

Evaluation of Recombinant Proteins-N-terminal Microsequencing and Detection of Residual Contaminants

利用Edman降解法,辅之以邻苯二甲醛阻断等技术,在蛋白/多肽测序仪-PTH氨基酸分析仪上微量自动测定了在大肠杆菌系统获得高效表达的重组人γ干扰素、α 2干扰素和白细胞介素-2等多种基因工程产品的N末端氨基酸序列。根据氨基酸色谱分析的原始资料,对上述蛋白质样品的N末端均一性进行了细致分析。在此基础上,借助序列库检索比较等计算机方法检定了这些样品中可能残留的痕量杂质蛋白,并对分析痕量残留蛋白的一般方法进行了讨论。分析结果表明重组人γ干扰素N末端均一;而α 2干扰素和白细胞介素-2的N末端不均一,其中主要含大致等量的N末端带有蛋氨酸与不带蛋氨酸的两种分子,仅在个别送检样品中发现痕量N末端丢失一个氨基酸的分子。通过在序列库中检索所测出的一段八肽疑似序列,推断在一批送检样品中含有痕量的卵清溶菌酶,经进一步纯化样品并测序后得以证实。本文结果为进一步优化和确定有关产品的生产流程并建立产品质量控制的标准提供了重要依据。

The N terminal amino acid sequence of purified recombinant humaninterferon-γ, interferon-α 2a and interleukin-2 expressed in E. coliwas determined on Applied Biosystems 477A Protein/Peptide Sequen erand 120A PTH Amino Acid Analyzer. From the raw chromatograpicdata of these samples, the identity and heterogeneity, the amount ofmethionine-plus species remaining in the final product, and the probableprocess contaminants were evaluated with the help of computer methodsincluding database searching. General methods to characterize trace con-taminants in protein sample were also discussed. Among the sequencedsamples, only interferon-γ was shown to be N-terminal homogenous.Methionine-containing species were found in interleukin-2 and inter-feron-α 2a. Chicken eggwhite lysozyme was detected at very small amountin one batch of sample. The results provide valuable information forthe development and improvement of preparation methods as well as re-gulatory responses to recombinant products.