PROKARYOTIC EXPRESSION AND PARTIAL CHARACTERIZATION OF MAIZE CHLOROPLAST FERREDOXIN 1
The ferredoxin (Fd) proteins in plant chloroplasts play important roles in cellular metabolism by delivering reducing equivalents through the [2Fe-2S]cluster in the active site to various essential oxido-reductive pathways.In this study, the chloroplast leading peptides from five Fd proteins of maize share the low homogeneity, whereas the mature Fd proteins deleted the leading peptides are high homogeneous, as displayed by the amino acid sequence alignments.The gene encoding mature maize ferredoxin 1 (Fdl) was cloned by RT-PCR using the total RNA from young leaves as the template.The cloned gene was inserted into pQE80 and p28SUMO plasmid, and transformed into Escherchia coli BL21 (DE3) respectively.The expressed Fdl fused with the histidine-tag (His-Fdl) or HisSUMO tag (HisSUMO-Fdl)at N terminus, was purified by Ni-NTA affinity chromatography independently.The recombinant Fdl protein was obtained by removing the HisSUMO using the specific protease Ulp.SDS-PAGE analysis showed that purified His-Fdl has a molecular mass of about 12kD.The purified HisSUMO-Fd1 has the absorption peaks at 315, 415 and 459 nm identified by UV-visible spectra scanning, and the [2Fe-2S]cluster determined by electron paramagnetic resonance (EPR) experiments.Several proteins from soluble extracts of young maize leaves were bound by the immobilized His Fd1, as shown by SDS-PAGE analysis.
国家科技期刊平台
NSTL
万方数据
维普
