The Preparation Of Anti-?₂ Microglobulin Nanobody 83F By E.coli

?₂-microglobulin??₂M?cannot be normally metabolized in patients with chronic renal failure.Excessive accumulation of?₂M can cause amyloidosis.Currently,hemodialysis cannot remove excess?₂M.Nanobodies?Nbs?have important value in immunosorption therapy based on its distinctive advantages such as miniaturized capture agent,high affinity,strong immunity and easy fusion expression.Therefore,it has great clinical significance to prepare a large number of anti-?₂M nanobodies.E.coli is considered as the preferred strategy for preparing nanobodies because of its low cost of cultivation and simple operation process.Anti-?₂M nanobody 83F has high affinity and strong binding specificity for?₂M,but preliminary studies have found that it is mainly expressed in the form of inclusion bodies.In order to obtain more active 83F,its expression by different hosts and fusion with different tags and conditions of inclusion renaturation were optimized in this paper.The specific research contents are as follows:?1?The preparation and expression of anti-?₂M nanobody-83F:First,the gene sequence of anti-?₂M nanobody-83F was cloned into pET29a,pET32a,pET39b,pET40b,pET41a and pET28a by gene recombination technolog.Then the recombinant plasmids were transformed into E.coli Shuffle T7?DE3?,E.coli Origami ^TM2?DE3?and E.coli BL21?DE3?,all strains stably expressed 83F.By optimizing the expression conditions,it was found that the expression levels in E.coli Shuffle T7?DE3?and E.coli BL21?DE3?were significantly better than those in E.coli Origami^TM2?DE3?;adding fusion tags could increase the expression of nanobody-83F;the expression levels at induced temperature 37°C were better than those at18°C.?2?Soluble analysis of anti-?₂M nanobody-83F:Nanobody-83F was induced at a low temperature of 18°C,resulting in more soluble protein.The highest soluble expression appeared when it was expressed by Shuffle T7?DE3?using pET39a?S39?and pET40b?S40?as vector,accounting for 14%and 16%of the supernatant proteins.After purification by metal chelate chromatography,about 62 mg and 77 mg recombinant proteins,at purity of93%and 95%respectively,were obtained from in per liter of TB medium.Moreover,the purified S39 and S40 had antigen-binding activity with an affinity constant as high as 1.1318×10^-6 M and 3.457×10^-7 M,respectively.After the fusion tags of S40 was excisioned,SPR assay showed that the affinity constant of 83F was 2.529×10^-7M,the affinity of the nanobody before and after excision of the tag approaches.?3?Inclusion body renaturation of anti-?₂M nanobody-83F:The highest inclusion body expression yield were obtained when 83F was expressed at 37°C by BL21?DE3?using pET29a as vector,accounted for 62%of the precipitated proteins.To refolding 83F inclusion body,a method of diluted renaturation was used after optimizing the renaturation conditions,the most appropriate renaturation condition was that:initial protein concentration was at 0.4mg/ml,droplet speed of 20 mM Tris-Hcl renaturation buffer?pH 8.0?containing 200 mM arginine,GSH:GSSH=1:2,12.5 mM,2,6-Dimethyl-?-cyclodextrin at 3 ml/min,placed at 4°C for 16 h.Finally,1221 mg refolded nanobody 83F can be obtained from 1 L TB medium with a purity of 95%.Measurement of circular dichroism spectroscopy demonstrated that the addition of suitable additives can facilitate formation of the correct secondary structure of the nanobody.Moreover,placed at 4°C for 16 h,the secondary structure and equilibrium dissociation constants of the renatured protein is similar to the proteins expressed in the supernatant,indicating that the renatured protein has high antigen-binding activity and has the correct secondary conformation.In conclusion,the preparation of the anti-?₂M nanobody 83F by E.coli was successfully obtained.On the one hand,the total expression and soluble expression of the nanobody 83F could be increased by adding fusion tags.On the other hand,by optimizing the conditions of diluted renaturation,inclusion bodies were successfully renatured.However,a comparative analysis found that the method of renaturation of inclusion bodies is less expensive for the preparation of nanobody 83F.