Wetzel R, Perry L J, Veilleux C
Biomolecular Chemistry Department, Genentech, Inc., South San Fransico, CA 94080.
Biotechnology (N Y). 1991 Aug;9(8):731-7. doi: 10.1038/nbt0891-731.
High level expression of the gene for human interferon-gamma (HuIFN-gamma) in E. coli JM101 cultured at 37 degrees C results in the distribution of over 90 percent of the total accumulated gene product into inclusion bodies (IBs). We have identified mutations throughout the molecule that alter the distribution between the soluble and inclusion body fractions without greatly affecting total expression level. Some mutants retain high biological activity but are localized almost entirely in the soluble fraction. Mutations affecting IB distribution as well as stability to intracellular proteolysis were detected by immunochemical screens and verified by gel assays. Immunochemical screens such as those employed here may allow identification of folding and stability mutants in heterologously expressed proteins when there is no other basis for selection or screening. These results also suggest that one solution to production problems arising from IB formation may be to identify mutations in the target protein that favor expression of soluble protein while retaining biological activity.
在37摄氏度培养的大肠杆菌JM101中，人γ干扰素（HuIFN-γ）基因的高水平表达导致超过90%的总积累基因产物分布在包涵体（IBs）中。我们已经在整个分子中鉴定出突变，这些突变改变了可溶性部分和包涵体部分之间的分布，而不会对总表达水平产生太大影响。一些突变体保留了高生物活性，但几乎完全定位于可溶性部分。通过免疫化学筛选检测影响IB分布以及对细胞内蛋白水解稳定性的突变，并通过凝胶分析进行验证。当没有其他选择或筛选依据时，此处采用的免疫化学筛选可能允许鉴定异源表达蛋白中的折叠和稳定性突变体。这些结果还表明，解决由IB形成引起的生产问题的一种方法可能是在靶蛋白中鉴定有利于可溶性蛋白表达同时保留生物活性的突变。
