Cloning and characterization of serpin from red king crab Paralithodes camtschaticus.

Serpins are a family of serine protease inhibitors that are involved in numerous physiological processes and are known to regulate innate immunity pathways. To advance our understanding of their role in P. camtschaticus, a commercially significant species, we cloned and characterized a serpin from this species, designated serpin PC, that has anticoagulant and anticomplement effects on human blood. We found that serpin PC is a secreted protein with a typical serpin-like primary structure that is similar to other known crustacean serpins. Recombinant serpin PC was found to have inhibitory activity against R/K-specific bovine cationic trypsin. The reaction proceeds through the formation of a stable covalent complex of peptidase with P1 residue R383 of serpin PC. This interaction is characterized by a relatively high overall inhibition constant kass=(2.3 ±â€¯0.7) × 106 M-1s-1 and an SI of 4.7 ±â€¯0.8. Protein localization by western blotting showed that serpin PC is present in the muscles and, to a lesser extent, the heart, whereas it is transcribed predominantly in hemocytes and the heart. Through peptidase activity profiling of hemocytes and plasma, we found that serpin PC inhibits at least two R/K-specific activities and showed that it inhibits phenoloxidase (PO) activity induction in hemocytes.

Genetic Engineering of Native Chain Combinations of B-Cell Repertoires on the Surface of Methylotrophic Yeasts Pichia pastoris.

We designed genetic constructs for exposing Fab-fragment library of natively paired single cell B-cell receptors on the surface of Pichia pastoris yeast cells. We have previously obtained the A17 antibody in our laboratory [6]. In this study we showed that the newly designed genetic constructs provide a compatible level of A17 antibody Fab fragment on the surface of yeast cells as well as in the case of vectors containing DNA fragments corresponding to each chain of the antibody. The data suggest that the developed approach for constructing immunoglobulin gene libraries is adequate and fully convenient for studying properties of the real human B-lymphocyte repertoire.