Figure 8.

Representative detail of vector construction and sPLA₂ fusion. Initially, pcDNA3.1 was modified by inserting a kappa secretory signal followed by a 6xHis tag and a modified multiple cloning site via a HindIII/XbaI double digestion. The resulting vector (A) was used directly to create 6xHis-tagged sPLA₂ constructs. In order to generate the various SUMO fusions, vector A was digested with Esp3I and ligated to Eco31I/XbaI digested, PCR-amplified, SUMO derivatives containing a new multiple cloning site (B) to give vector C. SUMO vectors (C) were digested with Esp3I and fused with sPLA₂ PCR products (D) digested with Eco31I, Esp3I, or BpiI. This strategy resulted in the creation of in-frame fusions (E) between the various SUMOs and sPLA₂s.