Screening Proteins Interacting With Sulfide: Quinone Oxidoreductase (sqr) Of Urechis Unicinctus

Sulfide is a toxin for most organisms which main poisoning effect is the inhibition to cytochrome oxidase, leading to impaired aerobic energy metabolism. In recent years, a number of coastal benthic organisms were found to oxidize sulfide to low toxic or nontoxic products to reduce or eliminate the sulfide toxicity on living organisms. Sulfide:quinone oxidoreductase (SQR) is considered to be a key enzyme in the oxidation process. In this paper, potential SQR interacting proteins from Urechis unicinctus, a echiuran worm distributed in China coast, were screened and initial analysed, providing the basic data for further investigation in oxidative metabolic mechanism.Prokaryocyte expression vector (pET28a(+)/SQR) was constructed by cloned U. unicinctus SQR open reading frame cDNA into pET28a(+), and then transformed into Escherichia coli BL21 (DE3) to get the recombinant protein. The 6His-SQR was expressed in the form of inclusion body in E. coli with the induction of IPTG. Dilution refolding was used to get the activated 6His-SQR which exhibited specific activity at 5.04μM/min/mg. Two probable 6His-SQR binding proteins, which show molecular weight of 40 ku and 60 ku, respectively, were screened from 50μM sulfide treatment U. unicinctus body wall total protein extraction solution by refolded 6His-SQR saturation binded to Ni2+-NTA resin (1mg Ni2+-NTA resin saturation binding 3mg 6His-SQR protein) using His-pulldown technique; The two protein was preliminary determined as cytochrome P450 (cytochrome P450)and adenosine triphosphate-binding cassette transporter (ATP-binding cassette transporter) by mass spectrometer analysis, furthmore, probable functions of the two proteins were also discussed.