Construction Of Of Two Types Of Immunoaffinity Chromatography Platforms Using Peptide-tag Specific Nanobodies As Affinity Ligands And Its Application

Affinity chromatography is generally regarded as a powerful tool that allows the purification of recombinant proteins with satisfactory purity in one single step.However,for most protein products,affinity purification methods for industrial applications are not readily available,mainly due to the lack of specific and robust natural counterparts that could function as affinity ligands.The development of camelid heavy chain antibodies(hcAbs)opened a new avenue for immunoaffinity applications.The single-domain antibody,also referred as nanobody or VHH,is known as the smallest antibody fragment that could maintain antigen-binding activity.Two typical nanobodies(BC2-nb and Syn2-nb)that are capable of specifically recognizing certain peptide-tag,were prepared through prokaryotic expression and proved to be able to bind their corresponding tag-fused enhanced green fluorescent protein(eGFP)in the nanomolar range affinity.In this study,we explored the applicability of nanobody-based peptide-tag immunorecognition systems as a platform for affinity chromatography.(1)DNA fragments of BC2-nb,eGFP-BC2T,Syn2-nb and eGFP-EPEA were synthesized from Genewiz(Suzhou,China)with codons being optimized for E.coli expression system.The above recombinant proteins were also engineered to have a hexahistidine tag for purification.BC2-nb(lane 1)and Syn2-nb(lane 3)could have good soluble expression in E.coli SHuffle T7 strain.BC2-nb displayed binding affinity in the low nanomolar range for binding eGFP-BC2T(1.4 nM),while the K_D of Syn2-nb in binding its target fusion tag was determined to be62.6 nM.(2)Through epoxy-based immobilization reaction,the two nanobodies were coupled on Sepharose CL-6B matrix under the same conditions.Sepharose CL-6B was activated with 1,4-butanediol diglycidyl ether in advance to have an epoxy density of 14μmol/mL.The immobilization amount of BC2-nb was measured to be 11.5 mg/mL moist resin,indicating a couping ratio of 38.3%,while the immobilization amount of Syn2-nb was 7.0 mg/mL moist resin,with the coupling ratio of 23.3%.(3)The immobilized affinity ligands exhibited high binding specificity toward their respective target proteins with the purity up to more than 90%directly from crude bacterial lysates in one single chromatographic step.they completely lost their binding abilities after 40and 18 cycles of use for BC2-nb resin and Syn2-nb resin,respectively.Therefore,nanobody-based affinity chromatography methods can be expected to be a poteintial complement to IMAC or the existing immunoaffinity chromatography.However,less efficient product recovery caused by excessive binding affinity is still a primary issue that should be addressed before nanobody-based affinity chromatography can act as a general platform for recombinant protein purification.