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 Synechococcus PCC 7002 which reacts with a conformational epitope of the PSI complex. As seen by immunoelectron microscopy, the mAb bound to the stromal side of the thylakoid membranes. The DNA sequence encoding variable regions of the mAb was cloned into recombinant plasmids, sequenced and expressed in Escherichia coli. ELISA, Western blots and immunoelectron microscopy provided evidence that the expressed paired variable domain (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 complex 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 antenna size of the isolated PSI/Fv was 139 +/- 9 chlorophyll a/primary electron donor. Flash-induced absorption-change measurements showed that the complex exhibited electron transfer from the primary electron donor, P700, to the Fe-S center, FA/FB. The position of the bound Fv fragment on the trimeric PSI surface was determined by high-resolution electron microscopy and digital image processing.
Tsiotis
G
G
Max-Planck-Institut für Biophysik, Frankfurt am Main, Germany.
Haase
W
W
Engel
A
A
Michel
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000737:chemistry* / Q000648:ultrastructure; D018076:DNA, Complementary; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics; D045342:Light-Harvesting Protein Complexes; D051379:Mice; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D045331:Photosystem I Protein Complex; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013057:Spectrum Analysis
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D007128:Immunoglobulin Fragments; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D011994:Recombinant Proteins; C055966:immunoglobulin Fv