Preparation Of An Immunoadsorbent Site-specific Immobilized With ?₂-microglobulin VHHs Via Formylglycine-generating Enzyme

Dialysis-related amyloidosis?DRA?,which has been widely recognized to be associated with the accumulation of?₂-microglobulin??₂m?in blood,is one of the most common complications in patients receiving long-term hemodialysis treatments.?₂m usually accumulates in the blood and joints of patients with advanced kidney disease and is generally believed to be a vital biomarker for the diagnosis and assessment of chronic kidney disease and its complications.Traditional hemodialysis devices based on hydrophobic interactions failed to eliminating?₂m from blood.Monoclonal antibody and scFv based immunoadsorbents were fabricated recently for the extracorporeal removal of?₂m from blood,and it could also prevent the significant loss of valuable proteins which are conducive to patients.Notwithstanding,the low stability and the high cost of the synthetic affinity ligands limit the development of these antibody-based immunoadsorbents.Single domain antibodies?also refered as VHHs?discovered recently possess high affinity,high stability and low immunogenicity.Here we present a facile method for the preparation of a VHH-based immunoadsorbent using the formylglycine-generating enzyme to introduce a site-specific aldehyde group which facilitates the subsequent immobilization at the C-terminus of the VHH.The novel sorbent exhibited a high specificity to?₂m.Meanwhile,combinatorial approaches based on VHH antibodies have permitted a rapid,feasible and low-cost synthesis of new synthetic affinity ligands of precise use with scale up potential in novel immunoadsorbent preparation.Conclusions were followed:?1?Expression of recombinant VHHs and FGE.The sequence of an anti-?₂m VHH antibody which contains a His-tag and an Ald-tag at C-terminal was inserted into a pET21a plasmid and expressed in E.coli shuffle?T7?.The yield of recombinant VHHs could reached to 400 mg/L.The purity of soluble VHHs was about 60%after the acid-assisted precipitation of endogenous proteins,which was really conducive to the subsequent one-step immobilization process.On the other hand,FGE was expressed and purified by metal ion affinity chromatography.The final yield could reach up to 300 mg/L.?2?The optimization of the catalytic process.The purified VHH was incubated with FGE at a molar ratio of 20:1 at 20?for 4 h.With the addition of 1 mM DTT in catalytic buffer,a maximum catalytic efficiency of 90%could be obtained.The binding affinity before and after catalysis measured by SPR?Biacore T200?was 1.220×10^-77 M and 2.308×10^-77 M,separately.These results showed that the VHH antibody possessed a high affinity and was capable to be used as ligands in the preparation of the immunoadsorbent.The supernatant was treated under an acetic acid-assisted precipitation procedure and then incubated with purified FGE.The conversion efficiency after optimization could reach up to 79%.The result suggested that VHH was possible to be immobilized directly onto agarose beads from the crude cell extract.?3?Preparation and evaluation of the VHH-based immunoadsorbent.The Schiff base reaction achieved higher efficiency than the hydrazone ligation in the immobilization of VHHs onto agarose beads.Under optimized catalytic conditions,the purified VHH could achieve a binding density of 2.0 mg VHH per mL gel and the sorbent exhibited an adsorption site density of 0.89 mg?₂m per mL settled gel.The VHHs modified by FGE had also been directly ligated to amino-activated beads from the crude cell extract with one-step method and the sorbent exhibited an adsorption site density of 0.75 mg?₂m per mL settled gel.It also demonstrated that this VHH-based adsorbent was specific to human?₂m in preliminary specificity experiments.