FUNCTIONAL DISPLAY AND EXPRESSION OF CHICKEN CYSTATIN USING A PHAGEMID SYSTEM

A recombinant phage antibody system has been used to display functiona lly active chicken cystatin, a small protein-type inhibitor of papain- like cysteine proteinases. A synthetic gene, AEF-[S1M, M29I, M89L] chi cken cystatin, was ligated into phagemid pCANTAB 5E, cloned, sequenced and displayed on the tips of filamentous phage as a fusion protein wi th protein III (gpIII). The gene was expressed also as soluble inhibit or in a non-suppressor E. coli strain. Recombinant phages were selecte d by binding to carboxymethylated-papain. The positive phages showed b inding to papain in dose dependent manner. The chicken cystatin-gpIII fusion protein was detected with anti-chicken cystatin antibody and wi th anti-protein III antibody by western blot analysis. Unfused soluble inhibitor was identified in the periplasma and showed strong papain i nhibitory activity. The results demonstrate that functionally active c ystatin can be displayed on the phage surface. This technology can be employed further to select inhibitor variants which differ in their bi nding affinity to papain and papain-like enzymes. (C) 1995 Academic Pr ess, Inc.