Figure 4

Test of CR fusion protein production. (A) The sequence spanning the C-REase junction has properties that might result in production of some free C protein. GCAAAAA has been associated with −1 ribosomal frameshifts (see text for references), and this would result here in termination at a nearby TGA triplet. (B) Production of NsoJS138I C-REase fusion protein, with an amino-terminal (upper) or carboxyl-terminal (lower) His₆ tag, was induced using a T7 RNA polymerase-dependent promoter (see Methods). The upper panels show the results from clones having a small carboxyl-terminal deletion (done in case the REase activity proved to be toxic), while the lower panels show full-length clones. Centrifugally-clarified whole-cell extracts were passed over affinity columns to purify the His-tagged polypeptides, and resolved on duplicate 10-20% gradient acrylamide SDS gels. For the lower panels, the extracts were prepared in the presence of protease inhibitors. One gel of each pair was stained (left), the other was electroblotted and probed with anti-His-tag antiserum (see Methods). Loaded amounts of protein per lane were 2.0 μg (upper), and 3.4, 6.8 and 5.1 μg (lower, left to right).