Protease Deficient Cho Cell Lines To Prevent Proteolysis of Recombinant Proteins

Researchers in Phil Berman's group at UC Santa Cruz have identified complement Component 1s (C1s) as the enzyme responsible for gp120 cleavage in CHO cells. C1s is an enzyme in the immune system that is responsible for cutting proteins at specific places to combat bacteria and viruses. Problematically, CHO cells also produce C1s that, in this case, proteolyzed the gp120 product.  

The team used CRISPR/Cas9 gene editing to create C1s-deficient CHO cell lines for gp120 expression. These new cells no longer make the C1s enzyme and thus, proteolysis does not occur. Recombinant gp120 expressed in these cells remain intact and antigenically correct, preserving the structure required for recognition by the neutralizing antibodies. The C1s-deficient CHO cells grew just as well as unaltered cells and produced similar yields. This is notable as the removal of C1s did not harm the productivity or viability of the cell line, making it suitable for large-scale manufacturing. 

They also removed another gene, MGAT1, which is responsible for the addition of glycans to proteins, called glycosylation. In HIV gp120, the glycan mannose-5 (Man5) is crucial for the recognition of gp120 by the broadly neutralizing monoclonal antibodies (bN-mAbs). These antibody targets are usually obfoscated by MGAT1 and thus, the MGAT1-deficient CHO cells, with simplified sugars, expose the antibody targets. These cells make a fully intact, properly glycosylated gp120 for use in a potential HIV vaccine.