Cloning And Heterologous Expression Of Auracyanin Gene From Phototrophic Bacterium

Microbial mats in hot springs are generally formed by phototrophic bacteria. These mat-forming thermophiles comprise oxygenic cyanobacteria and the so-called filamentous anoxygenic phototrophs (FAPs) represented by model species Chlorqflexus aurantiacus. These groups of bacteria contain one type of blue copper proteins called auracyanins. They function as electron carriers in those organisms which lack soluble cytochromes as electron-transfer agents. In model species, there are two forms that have been discovered, A and B. The previous studies suggest they have one very similar globular domain but distinct N-terminal, indicating differences in locations and functions.In one recently discovered FAPs Roseiflexus castenholzii genomic sequence analysis showed that this species has only one auracyanin, while Chloroflexus aurantiacus is known to have two auracyanins. Thus, the question of what is the function mechanism and the modality of this copper binding protein still needs to be answered.The topic of this study is to use genetic engineering bacteria to clone and overexpress auracyanin in order to investigate the similarity and difference of the full-length vs. soluble part of the protein in the electron transfer process. The gene bcd encoding full-length protein and the gene bed2encoding soluble Cu-containing domain were amplified by PCR with extracted genomic DNA as model. The expression vector pPAL7harboring bed and bcd2gene respectively, were transformed into.E.coliBL21(DE3) to yield the recombinant strain E coli (pFAL7-bcd) and E coli (pPAL7-bcd2). The recombinant E. coli(pPAL7-bcd2) produced6.6mg/ml protein under the induction of1.0mmol/L IPTG at37℃for5h; at similar conditions, E.coli(pPAL7-bcd) produced7.0mg/ml protein. Havested cells were lysised by sonication. The total lysate is divided to solubiiv supemantant and pellet part by high speed centrifugation. The recombinaat proteins are located in cytosol of E. coli based on Tricine-SDS-PAGE analysis. Then, the target proteins were purified with the Bio-Scale Mini Cartridge affinity purifications system. The purified sample was analysl. by Tricine-SDS-PAGE and Western blotting analysis. The soluble part of protein is of high parity and used for antibody production for future work. The three proteins are analysed by MALDI-TOF mass spectroscopy, absorption and electron paramagnetic resonace specrosce (?) identify the molecular feature of auracyanins after reconstituted with copper ion.The soluble and full-length aurayanin from Roseiflexus were compared to the countpart (?) model species Chloroflexus. Our results together suggest that auracyanin from from Roseiflexus shares features with each of auracyanin A and B. Amino acid sequence alignment of auracyanins from FAPs also demonstrated the chimeral feature of the primary structure of the Roseiflexus auracyanin, i.e., auracyanin A-like amino-terminal characteristics and auracyanin B-like one-residue spacing at the Cu-binding loop in the carboxyl-terminus.