Purification And Optimization Of Crystallize Conditions For Cytochrome B₆f Complex From Spinach

Cytochrome b₆f protein complex （of Cytochrome b₆f complex complex, referred to asthe Cyt b₆f complex） is one of the photosynthesis primary light energy conversion processof four major membrane protein supramolecular complexes. It connects the reactioncenters of photosystems I and II linear electron transport and cyclic electron transportaround PS I.This electron transfer process is coupled to proton translocation across themembrane that generates a transmembrane electrochemical proton gradient used tosynthesize adenosine triphosphate （ATP）. Therefore, Cyt b₆f plays a very important role inthe light energy transducing process of photosynthesis. Although High-resolution X-raycrystallographic structures of the Cyt b₆f from Bacteria and Chlamydomonas reinhardtiihave been got in2003, the crystal structure of Cyt b₆f from the higher plants has not yetreceived. In recent years, laboratories around the world are trying to get the crystalstructure of Cyt b₆f from the higher plants as early as possible, to further reveal thephysiological function of Cyt b₆f protein complex of Chl a and β-Car molecules, whichfurther clarify the photosyntheticrole efficiently transduce the molecular mechanism of itsregulation is a very important significance.Order to get crystal structure of Cyt b₆f from higher plants, we use spinach as astarting material, to do a lot of improvements on the basis of the original separation andextraction process. The improved method allows us first time to isolated and purified Cytb₆f from spinach thylakoid membranes could be used to crystal, which made us to getpreliminary crystals of Cyt b₆f. Cyt b₆f protein crystal size of the state currently is notenough for data collection, but the experimental basis for further optimization andanalytical studies of higher plants Cyt b₆f protein crystallization.The main improvements are as follows:（1） The NaBr washing step was repeated to completely remove the extrinsic protein（2） Simplify the process of the two ammonium sulfate fractionation preparation forone ammonium sulfate fractionation preparation（3） Optimize the extraction conditions（4） To optimize the conditions of sucrose density gradient centrifugation.（5） Using PEG4000precipitation method for concentration, the protein was furtherpurified.