Separation And Purification Of Cellodextrin Transporter CDT-1 And CDT-2

Cellodextrin transporter CDT-1 and CDT-2 is of high value for yeast engineering for biorefinery and bio-product production.CDT-1 and CDT-2 are required to induce the expression and secretion of cellulases in filamentous fungi such as Neurospora crassa.Previous studies showed that CDT-1 and CDT-2 may act as transporting receptors of cellodextrin for the induction of cellulases in N.crassa,but whether and how CDT-1 and CDT-2 mediated cellodextrin signaling is still unclear.Separating CDT-1 and CDT-2 and identifying the modification and quaternary structure plays a critical role for understanding structure and function relashionships of CDT-1 and CDT-2 and helps exploring the mechanisms underlying the induction and expression of cellulases and conducting direct evolution of CDT-1 and CDT-2 for improved cellodextrin uptake.As a consequence,seperation and purification of the membrane protein CDT-1 and CDT-2 is crucial to address the above issue.Here,we are aiming to establish a method for seperation and purification of CDT-1 and CDT-2 from N.crassa.First,we constructed the strains,cdt-1::gfp::bccd and cdt-2::gfp::bccd,in which cdt-1and cdt-2 were fused with gfp and biotin-binding doman bccd.With the tag GFP::BCCD,we confirmed that crystalline cellulose(Avicel)can effectively induce the expression of CDT-1::GFP::BCCD and CDT-2::GFP::BCCD and the constructed BCCD-fusion proteins were efficiently biotinylated in vivo.Triton X-100,CHAPS,Tween-20 and digitonin were shown to be suitable for solubilizing CDT-1::GFP::BCCD,and the mobility of CDT-1::GFP::BCCD varied when different detergents,such as tween-20 and digitonin,were applied,indicating changes of conformaton or oligomerization state of CDT-1::GFP::BCCD.Chemical crosslinker A was applied to stablize CDT-1::GFP::BCCD or CDT-2::GFP::BCCD oligomers or complexes.After crosslinking,stablized oligomers or complexes of CDT-1::GFP::BCCD or CDT-2::GFP::BCCD was detected.Upon treatment with Avicel and sucrose,respectively,during growth,the resulting CDT-2::GFP::BCCD oligomers or complexes showed significantly different mobility on BN-PAGE,suggesting that the state of CDT-2::GFP::BCCD oligomers or complexes changes in response to cellulose and sucrose.Pulldown experiments showed that CDT-1::GFP::BCCD or CDT-2::GFP::BCCD oligomers or complexes were able to be effectively captured by Dynabeads M280~?Streptavidin.In conclusion,we successfully worked out the conditions for crosslinking,solubilizing,separating and purifying the oligomers or complexes of CDT-1::GFP::BCCD and CDT-2::GFP::BCCD,respectively.This work lays a foundation for our further analysis of the structure and function of CDT-1 and CDT-2 oligomers or complexes,including their post-translational modifications,compositions and structures of their oligomers or complexes.This work will also provide a method for studying the relationship of the structure and function of other sugar transpoters,and help for further exploiting the way to make use of these transpoters for improving economic benefit.