ISOLATION AND STRUCTURAL CHARACTERIZATION OF TRIMERIC CYANOBACTERIAL PHOTOSYSTEM-I COMPLEX WITH THE HELP OF RECOMBINANT ANTIBODY FRAGMENTS

A monoclonal antibody was derived from mice immunized with the native trimeric, photosystem I (PSI) complex from the cyanobacterium Synechoc occus PCC 7002 which reacts with a conformational epitope of the PSI c omplex. As seen by immunoelectron microscopy, the mAb bound to the str omal side of the thylakoid membranes. The DNA sequence encoding variab le regions of the mAb was cloned into recombinant plasmids, sequenced and expressed in Escherichia coli. ELISA, Western blots and immunoelec tron microscopy provided evidence that the expressed paired variable d omain (Fv) fragments bind to the antigen in the same way as the parent mAb. A one-step purification was applied to purify the trimeric PSI c omplex using an affinity tag attached to the Fv fragment. Analysis by gel electrophoresis and N-terminal sequencing revealed the presence of the psaA, psaB, psaC, psaD, psaE, psaF and psaL gene products. The an tenna size of the isolated PSI/Fv was 139+/-9 chlorophyll a/primary el ectron donor. Flash-induced absorption-change measurements showed that the complex exhibited electron transfer from the primary electron don or, P-700, to the Fe-S center, F-A/F-B. The position of the bound Fv f ragment on the trimeric PSI surface was determined by high-resolution electron microscopy and digital image processing.