Mutations in Human Interferon Gamma Affecting Inclusion Body Formation Identified by a General Immunochemical Screen

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.