Co-localization Of Proteins With Defined Sequential Order And Controlled Stoichiometric Ratio On Magnetic Nanoparticles

Multiple enzymes are usally needed to work together in a cascade with different ratios while researches on co-immobilization of enzymes were few,especially for immobilizing as a certain sequence and proportion.Herein,we developed a novel method to immobilize multiple enzymes on nanoparticles with a defined sequential order and a controlled stoichiometric ratio.We choosed core/shell magnetic nanoparticles as the support,one was Fe3O4/PVIM-Ni2+,the loading Ni2+ could be bonded to His tag;another was Fe3O4-Au,the au loaded on the surface could be combined with thiol in covalent binding form.We designed two kinds of scaffoldins:scaffoldins ? contain His tag,thiol and several olpB cohesins(1?7)which can be combined with docCipA,and they can be loaded on the nanoparticles,too.So we used the dockerin-fused fluorescent proteins[GFP(Green fluorescent protein)and DsRed(Discosoma sp.Red)]as models to study the immobilization.We found the loading capacity of scaffoldin-treated nanoparticles increased when the number of olpBs varied from one to four and decreased from four to seven(the max value was 0.937?mol/g).Scaffoldins II contain His tag and cohesins A and C,which can be combined with dockerins docA and docC respectively.We designed four Scaffoldins containing A and C with different numbers and secquences,which was in accordance with the immobilized fluorescent proteins.Scaffodins with more olpB were more difficult to express.Considered these factors,we choosed scaffoldins containing three cohesins(ACA and CAC)to research the co-immobilization on the nanoparticles.The results showed that flurescent proteins(GFP and DsRed)could be immobilized on nanoparticles with a defined sequential order decided on the sequence of cohesins and a controlled stoichiometric ratio varied from about 1:3 to 7:4.The maximum loading capacity could reach to?0.831 ?mol/g nanoparticles.