Limits...
Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp.

Wang R, Xiang S, Feng Y, Srinivas S, Zhang Y, Lin M, Wang S - Front Cell Infect Microbiol (2013)

Bottom Line: Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv.In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples.These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody.

View Article: PubMed Central - PubMed

Affiliation: The Ministry of Education Key Laboratory of Biopesticide and Chemical Biology, College of Life Sciences, Fujian Agriculture and Forestry University Fuzhou, China.

ABSTRACT
Single-chain variable fragment (scFv) is a class of engineered antibodies generated by the fusion of the heavy (VH) and light chains (VL) of immunoglobulins through a short polypeptide linker. ScFv play a critical role in therapy and diagnosis of human diseases, and may in fact also be developed into a potential diagnostic and/or therapeutic agent. However, the fact that current scFv antibodies have poor stability, low solubility, and affinity, seriously limits their diagnostic and clinical implication. Here we have developed four different expression vectors, and evaluated their abilities to express a soluble scFv protein. The solubility and binding activity of the purified proteins were determined using both SDS-PAGE and ELISA. Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv. In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples. These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody.

Show MeSH
Western blotting and affinity determination. (A) Western blotting analysis of the binding activity of scFv obtained by Skp co-expression to the TLH antigen. Left panel: SDS-PAGE results for the extracted TLH; Lane M: Protein molecular weight markers; Lanes 1, 2: extracted TLH. Right panel: Western blotting results; Lanes 3, 4: TLH band at 47 kDa bound by scFv obtained by Skp co-expression. (B) Specificity analysis of scFv obtained by Skp co-expression. BSA, KLH, and associated antigen proteins (VP1668 and YscF) of V. parahaemolyticus were coated on 96-well plates in triplicate (5 μ g/mL, 100 μ L/well). The purified scFv were added to the reaction wells and incubated for 2 h at 37°C. Specificity of Skp co-expressed scFv was determined using an anti-6×His tag antibody. (C) Affinity Determination of Skp co-expressed scFv. The affinity of anti-TLH Skp co-expressed scFv for the TLH antigen was studied by Microcalorimetry using the Nano ITC-LV. The measured data was used for the quantitative determination of the affinity constant of the anti-TLH Skp co-expressed scFv antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3818579&req=5

Figure 5: Western blotting and affinity determination. (A) Western blotting analysis of the binding activity of scFv obtained by Skp co-expression to the TLH antigen. Left panel: SDS-PAGE results for the extracted TLH; Lane M: Protein molecular weight markers; Lanes 1, 2: extracted TLH. Right panel: Western blotting results; Lanes 3, 4: TLH band at 47 kDa bound by scFv obtained by Skp co-expression. (B) Specificity analysis of scFv obtained by Skp co-expression. BSA, KLH, and associated antigen proteins (VP1668 and YscF) of V. parahaemolyticus were coated on 96-well plates in triplicate (5 μ g/mL, 100 μ L/well). The purified scFv were added to the reaction wells and incubated for 2 h at 37°C. Specificity of Skp co-expressed scFv was determined using an anti-6×His tag antibody. (C) Affinity Determination of Skp co-expressed scFv. The affinity of anti-TLH Skp co-expressed scFv for the TLH antigen was studied by Microcalorimetry using the Nano ITC-LV. The measured data was used for the quantitative determination of the affinity constant of the anti-TLH Skp co-expressed scFv antibody.

Mentions: As shown in Figure 5A, a clear band was revealed via western blotting (Figure 5A, lane 3, 4), and the result demonstrated that the TLH band at 47 kDa could be recognized by scFv obtained by Skp co-expression. To further determine the specificity of Skp co-expressed scFv protein to TLH antigen, ELISA was carried out. As shown in Figure 5B, Skp co-expressed scFv was found to be specific to TLH, while there was no cross binding to any other antigen-associated proteins such as BSA, KLH, Vp1668, and YscF. These results indicated that the co-expressed scFv could recognize TLH specifically and be used as an antibody reagent to detect TLH.


Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp.

Wang R, Xiang S, Feng Y, Srinivas S, Zhang Y, Lin M, Wang S - Front Cell Infect Microbiol (2013)

Western blotting and affinity determination. (A) Western blotting analysis of the binding activity of scFv obtained by Skp co-expression to the TLH antigen. Left panel: SDS-PAGE results for the extracted TLH; Lane M: Protein molecular weight markers; Lanes 1, 2: extracted TLH. Right panel: Western blotting results; Lanes 3, 4: TLH band at 47 kDa bound by scFv obtained by Skp co-expression. (B) Specificity analysis of scFv obtained by Skp co-expression. BSA, KLH, and associated antigen proteins (VP1668 and YscF) of V. parahaemolyticus were coated on 96-well plates in triplicate (5 μ g/mL, 100 μ L/well). The purified scFv were added to the reaction wells and incubated for 2 h at 37°C. Specificity of Skp co-expressed scFv was determined using an anti-6×His tag antibody. (C) Affinity Determination of Skp co-expressed scFv. The affinity of anti-TLH Skp co-expressed scFv for the TLH antigen was studied by Microcalorimetry using the Nano ITC-LV. The measured data was used for the quantitative determination of the affinity constant of the anti-TLH Skp co-expressed scFv antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3818579&req=5

Figure 5: Western blotting and affinity determination. (A) Western blotting analysis of the binding activity of scFv obtained by Skp co-expression to the TLH antigen. Left panel: SDS-PAGE results for the extracted TLH; Lane M: Protein molecular weight markers; Lanes 1, 2: extracted TLH. Right panel: Western blotting results; Lanes 3, 4: TLH band at 47 kDa bound by scFv obtained by Skp co-expression. (B) Specificity analysis of scFv obtained by Skp co-expression. BSA, KLH, and associated antigen proteins (VP1668 and YscF) of V. parahaemolyticus were coated on 96-well plates in triplicate (5 μ g/mL, 100 μ L/well). The purified scFv were added to the reaction wells and incubated for 2 h at 37°C. Specificity of Skp co-expressed scFv was determined using an anti-6×His tag antibody. (C) Affinity Determination of Skp co-expressed scFv. The affinity of anti-TLH Skp co-expressed scFv for the TLH antigen was studied by Microcalorimetry using the Nano ITC-LV. The measured data was used for the quantitative determination of the affinity constant of the anti-TLH Skp co-expressed scFv antibody.
Mentions: As shown in Figure 5A, a clear band was revealed via western blotting (Figure 5A, lane 3, 4), and the result demonstrated that the TLH band at 47 kDa could be recognized by scFv obtained by Skp co-expression. To further determine the specificity of Skp co-expressed scFv protein to TLH antigen, ELISA was carried out. As shown in Figure 5B, Skp co-expressed scFv was found to be specific to TLH, while there was no cross binding to any other antigen-associated proteins such as BSA, KLH, Vp1668, and YscF. These results indicated that the co-expressed scFv could recognize TLH specifically and be used as an antibody reagent to detect TLH.

Bottom Line: Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv.In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples.These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody.

View Article: PubMed Central - PubMed

Affiliation: The Ministry of Education Key Laboratory of Biopesticide and Chemical Biology, College of Life Sciences, Fujian Agriculture and Forestry University Fuzhou, China.

ABSTRACT
Single-chain variable fragment (scFv) is a class of engineered antibodies generated by the fusion of the heavy (VH) and light chains (VL) of immunoglobulins through a short polypeptide linker. ScFv play a critical role in therapy and diagnosis of human diseases, and may in fact also be developed into a potential diagnostic and/or therapeutic agent. However, the fact that current scFv antibodies have poor stability, low solubility, and affinity, seriously limits their diagnostic and clinical implication. Here we have developed four different expression vectors, and evaluated their abilities to express a soluble scFv protein. The solubility and binding activity of the purified proteins were determined using both SDS-PAGE and ELISA. Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv. In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples. These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody.

Show MeSH