Kosovský, J., Durmanová, V., Kúdelová, M., Rezuchová, I., Tkáciková, L. and Rajcáni, J.
Notes: The authors used the PinPoint™ Xa-1 Vector to clone two herpes simplex virus (HSV) genes and express the encoded proteins, IE63 and thymidine kinase, in E. coli JM109 for use as immunogens. Proteins expressed from the PinPint™ Vectors have a biotinylated tag, and the authors used this tag to purify the HSV proteins using the SoftLink™ Soft Release Avidin Resin. In a large-scale purification, the typical yield of IE63 protein was ~650µg/L. The recommended PinPoint™ purification protocol was modified in several ways: 1) the optimal incubation temperature and time for protein induction in E. coli cultures was chosen based on JM109 growth curves; 2) the post-induction time was optimized by monitoring protein expression levels using Western blot analysis; 3) the length of time that proteins were allowed to bind to the SoftLink™ Resin was optimized. The authors found that shortening the length of binding time to 2 hours eliminated the co-purification of endogenous biotinylated proteins in E. coli. (3850)