Design And Modification Of Antibody Based On GFP

Single-chain variable fragment (scFv) is a class of engineered antibodies generated by fusion of the heavy (VH) and light chains (VL) of immunoglobulin gene through a short polypeptide linker, and it is widely used as diagnostic and therapeutic agents or biosensors for a majority of human diseases. However, the limitations of the present scFv antibody in terms of stability, solubility and affinity are challenging to produce by the traditional antibody screening and expression formats. Hence, it is very important to develop a feasible approach for reducing these limitations.In this study, the superfolder GFP, a GFP variant that has high stability and improved folding kinetics, was used as a template for different regions of antibody (scFV, VH, VL, HCDR3and LCDR3) insertion at loop9. The binding activities of superfolder GFP-based antibodies were determined by ELISA. Compared to the traditional anti-TLH scFv antibody, the G9-LCDR3has the similar biding activity to TLH, with the binding activity being higher than all the others. Although the G9-HCDR3derived from HCDR3insertion reacted with TLH, but the binding activity is lower than G9-LCDR3and scFv. In fact, these proteins expressed from VL, VH and scFv insertions showed a very low binding activity to TLH.We were interested in determining whether the different CDR regions or the different length CDR3could affect the binding activity of antibody to recognize TLH antigen. Six different CDR regions or three different formats of HCDR3or LCDR3fragments were inserted into the same site of loop9as the former, and the binding activities of purified antibodies were analyzed by ELISA. ELISA result showed that amongst of six different proteins, G9-LCDR3a showed the highest binding activity to TLH in all the three LCDR insertions, while the G9-HCDR3a showed the highest binding activity in all the three HCDR insertions, and the G9-LCDR3a is the best one. The results derived from ELISA demonstrated that LCDR3played a key role in this antigen recognition. The purified G9-LCDR3a showed the highest binding activity in all the three LCDR3insertions. While there were no obvious differences in the binding activity of these three HCDR3insertions. In view of this result, LCDR3a and HCDR3a were regarded as the best formats for CDR3insertion, indicating that the shorter insertion of CDR3might be the best formats.To understand the interaction between G9-LCDR3/HCDR3and TLH antigen, all the amino acids of CDR3were mutated to alanine by site-directed mutagenesis for scanning the key amino acids that responsible for binding. Compared to the wild-type G9-LCDR3, the binding activities of the four mutated proteins (Q/A, R2/A) decreased greatly, showing the amino acids Q and R2of LCDR3were critical for the interaction between G9-LCDR3and TLH antigen. To further enhance binding activity of G9-LCDR3to TLH antigen, all the amino acids of CDR3insertion regions were mutated to lysine by site-directed mutagenesis. Compared to the wild-type G9-LCDR3insertion, the binding activity of the mutated G9-LCDR3-R2/K has increased greatly. On the basis of R2/K construct, the key amino acids Q (LCDR3) was also mutated to other seven different hydrophilic amino acids, compared to the G9-LCDR3-R2/K, the binding activity of all mutants decreased greatly, and this phenomenon implied that the amino acids Q is very important and immutable. To identity the versatility of this system, LCDR3/HCDR3regions from three different scFvs that are specific to three toxins (FBI, TTX and CIT), respectively, were inserted into loop9by the same format. These proteins were expressed and purified successfully, and the purified GFP-Abs also retains the binding activity to their antigen respectively. These results demonstrated that this system is feasible for generation a functional antibody based GFP scaffold.