Wetzel R, Perry L J, Veilleux C, Chang G
Department of Biomolecular Chemistry, Genentech, Inc., South San Francisco, CA 94080.
Protein Eng. 1990 Jul;3(7):611-23. doi: 10.1093/protein/3.7.611.
We have developed an expression/mutagenesis system and a series of screening procedures for the study of structure-function relationships in human interferon-gamma (HuIFN-gamma). Here we report a preliminary evaluation of the C-terminal portion of the molecule. An expression vector, p652trp gamma, was constructed which includes (i) the HuIFN-gamma gene under control of the trp promoter, (ii) elements controlling replication of both single- and double-stranded versions of the vector DNA; and (iii) the ampicillin resistance gene. (Other vectors using these same elements were constructed but proved to be unsatisfactory, being characterized by a rapid decline, as cells containing them were passaged, in their potential to achieve high expression levels.) A mutagenesis cassette was constructed by introduction of unique restriction sites flanking the nucleotides encoding the C-terminal 23 amino acids, and this cassette was replaced with chemically synthesized, degenerate oligonucleotides by ligation. Colonies from cells transformed with the reconstructed vector were stored in LB glycerol in microtiter plates, and these were screened by hybridization with synthetic oligonucleotides. Plates were grown in minimal medium to express the encoded interferon and lysed by an efficient, mild procedure. A polyclonal antibody specific for the C-terminal four amino acids of HuIFN-gamma was used to establish that the lysis procedure preserved the C-terminus, and to score for frame shift and nonsense mutations. Immunochemical assays also were used, with mixed results, to quantify IFN-gamma concentration in the lysate. An antiviral assay was employed to assess biological activity. Over 1000 isolates were screened and clones with properties representative of various classes of phenotypes were further characterized, in some cases after partial purification from the lysate. Three types of mutations were isolated: point mutations, nonsense mutations and frame shift mutations. The results from each type of mutation confirm earlier observations of the important role of basic residues in the 128-131 region of the molecule for biological activity. At the same time, the results suggest that most residues within the cassette can be altered without significant effects on biological activity. These results are discussed in the context of several possible mechanisms.
我们已开发出一种表达/诱变系统以及一系列筛选程序，用于研究人γ干扰素（HuIFN-γ）的结构-功能关系。在此，我们报告对该分子C末端部分的初步评估。构建了一个表达载体p652trpγ，其包含：（i）处于色氨酸启动子控制下的HuIFN-γ基因；（ii）控制载体DNA单链和双链形式复制的元件；以及（iii）氨苄青霉素抗性基因。（使用相同元件构建了其他载体，但被证明不令人满意，其特征是含有这些载体的细胞传代时，实现高表达水平的潜力迅速下降。）通过在编码C末端23个氨基酸的核苷酸两侧引入独特的限制性酶切位点构建了一个诱变盒，并用化学合成的简并寡核苷酸通过连接替换该诱变盒。用重组载体转化的细胞形成的菌落保存在微量滴定板的LB甘油中，并用合成寡核苷酸进行杂交筛选。平板在基本培养基中生长以表达编码的干扰素，并通过高效、温和的程序裂解。使用针对HuIFN-γ C末端四个氨基酸的多克隆抗体来确定裂解程序保留了C末端，并对移码突变和无义突变进行评分。免疫化学测定也用于定量裂解物中IFN-γ的浓度，结果不一。采用抗病毒测定来评估生物活性。筛选了1000多个分离株，具有代表各种表型类别的特性的克隆在某些情况下从裂解物中部分纯化后进一步进行表征。分离出三种类型的突变：点突变、无义突变和移码突变。每种类型突变的结果证实了早期观察到的分子128 - 131区域中碱性残基对生物活性的重要作用。同时，结果表明该诱变盒内的大多数残基可以改变而对生物活性无显著影响。在几种可能的机制背景下对这些结果进行了讨论。
