Intein-mediated Soluble Expression And Purification Of Nanobody By Escherichia Coli

Single-domain antibodies(sdAbs) with fully functional antigen-binding fragments arerecently cloned from the variable domain of heavy chain of heavy-chain antibody (VHH) ofcamelids. Their comparative small size, high stability and soluility, and ability to bindepitopes inaccessible for conventional antibodies making them especially suitable for manytherapeutic and biotechnological application. In spite of the fast technological development ofprotein expression, E.coli is still the most attractive host for rapid and cheap recombinantproteins production in scientific research and drug application. However, successfulexpression of souble and correctly folded protein is a frequently challenge.The developmentand application of fusion tags that can facilitate protein expression and solubility partly solvesthe problem. However, the fusin tags have to be removed by proteases in most case. Since thetag removal using proteases increases cost and introduces extra purification steps, it remains asignificant problem that must be solved. Intein has also been widely used in proteinproduction, self-cleaving of the intein fusion protein can be induced at the intein’s C-terminusor N-terminus and the cleavage produce the natural protein. The engineered self-cleavingintein exhibited rapid C-terminal cleaving properties in response to mild changes in pH andtemperature.In order to establishing a method for highly effective producing VHH, in this study, fourdifferent parts of work have been finished as follows:1. Construction of the expression vector with different fusion tagThe nanobodies of porcine circovirus type2(PCV2) Cap gene were amplified by PCR,then used as the target gene, three expression vector with different N-terminus fusion tags,PHSIE-VHH (His-Sumo-Intein), PHSIE-intein-VHH(His-Intein) and pET32a-intein-VHH(His-Trx-Intein) were constructed.2. Compared the effects of three N-terminus fusion tagsThree recombinant plasmids were transformed into E coil BL21(+) and induced by IPTG,respectively. the IPTG was added to a final concentration of0.4mmol/L and the culutureswere then continued for an additional6h at30℃or37℃. The SDS-PAGE electrophresisshowed that all tags enhanced the solubility and production, and the His-Trx-Intein is the beststraegy. 3. Intein-mediated VHH protein purificationRecombinat proteins were purified by Ni-NTA column chromatography, and tags wereremoved by Intein as self-cleavaged, different cleavage conditions were tested. The resultsshowed that the recombinant un-tagged VHH were released after the purifed proteins wereincubated at pH7.0and25℃for24h,. The released VHH was highly active, with theconcentration of about20μg/ml.4. Double antibody sandwich ELISA was applied to detect the activity of released VHH.The released VHH15-33protein was coated overnight at4℃on Maxisorp96-well plates;the plates were blocked with1%BSA-phosphate-buffered saline with0.05%Tween20(PBST) for1h at37℃, incubated with virus lysis for2h; then the6×His tag fused withVHH6-14fragments were loaded for2h at37℃; The wells were washed three times withPBST and treated with100μl of horseradish peroxidase(HRP)-Conjugated Anti His-TagAntibody at a1:4000dilution for1h at37℃. Finally, the reaction was developed byadding tetramethylbenzidine (TMB) and stopped by adding2NH2SO4, after30min ofincubation. The absorbance was measured in a microplate reader at450nm(Bio-Rad).The results indicated that the best coated concentration of VHH15-33protein was1:100andthe virus lysis was1:400; and proteins were significantly stronger than negative serumcontrol.which indicated that most of the selected VHHs interacted with PCV2viral lysis.