Modification And Exploration Of Recombinant Immunotoxin DAB389-4D5 ScFv With LOV2

Immunotoxin is a kind of anti-tumor drug that has been widely studied in recent years.It has the characteristics of targeting and killing ability to tumor cells.The LOV2 domain is derived from plant Phototropin.It undergoes conformational changes in assist of Flavin Mononucleotide(FMN)under 450-470nm blue light.The effect is precise and reversible,and now it has been widely used in the light regulation of bioengineered proteins and cellular processes.The split intein Npu DnaE is a common self-splicing element in bioengineering.Its self-splicing effect is accurate and rapid,and it is often used in protein purification and the regulation of biological processes.In this article,the split intein Npu DnaE InN-LOV2-InC(InLOV2)is inserted into the immunotoxin DAB389-4D5 scFv,to achieve the optical control of immunotoxin toxicity.InLOV2 was inserted into DAB389-4D5 scFv(D4InLOV)by fusion PCR.The recombinant expression strains are obtained and all have soluble expression.The results of MTT assay showed that the IC50 value of D4InLOV108 against SK-OV-3 was 0.75 nM under illumination,and 1.07 nM under dark incubation.The result shows effective optical-regulated inhibition of the recombinant protein.Meanwhile inhibition ability of D4InLOV108 against low-HER2-level H460 cells was significantly reduced with the inhibition rate of 27%,indicating its effective targeting to HER2.In order to realize better optical regulation,D4InLOV530 was chosen to be optimized.The insertion site is located in the linker between heavy and light chain of 4D5 scFv.Several amino acid residues have been deleted from the linker.The article uses MTT assay to calculate the inhibition rate.The results of MTT assay showed that the IC50 value of D4InLOV530LD5 against SK-OV-3 was 0.05 nM under illumination,and 0.09 nM under dark incubation,indicating the improved optically-regulated inhibition of recombinant protein.In this paper,the optical control element was successfully inserted into DAB389-4D5 scFv,and the optical regulation of anti-tumor activity was realized in some proteins.This article takes recombinant immunotoxin as the target of regulation.It is an innovative use of optical regulation elements to regulate protein functions.The article also provides ideas and experience for the precise regulation of immunotoxin.