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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.

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Protein purification and identification. (A) Protein purification. The purified protein was determined by SDS-PAGE. M: Molecular weight of marker proteins; Lane 1–4: the purified proteins for pACYC-Duet-scFv, pACYC-Duet-scFv-skp, pET28a-scFv, and pET32a-scFv, respectively. (B) Western blotting results. Lane 1–4: the detected bands for pACYC-Duet-scFv, pACYC-Duet-scFv-skp, pET28a-scFv, and pET32a-scFv, respectively. (C) ELISA. TLH antigen were coated on 96-well plates in triplicate (5 μ g/mL, 100 μ L/well), and the purified scFv proteins were added to the reaction wells after blocking and washing. The binding activities of the four purified proteins were determined using an anti-6×His tag antibody. (D) Quantitative result of ELISA*, P < 0.05, compared with control antibody treatment.
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Figure 4: Protein purification and identification. (A) Protein purification. The purified protein was determined by SDS-PAGE. M: Molecular weight of marker proteins; Lane 1–4: the purified proteins for pACYC-Duet-scFv, pACYC-Duet-scFv-skp, pET28a-scFv, and pET32a-scFv, respectively. (B) Western blotting results. Lane 1–4: the detected bands for pACYC-Duet-scFv, pACYC-Duet-scFv-skp, pET28a-scFv, and pET32a-scFv, respectively. (C) ELISA. TLH antigen were coated on 96-well plates in triplicate (5 μ g/mL, 100 μ L/well), and the purified scFv proteins were added to the reaction wells after blocking and washing. The binding activities of the four purified proteins were determined using an anti-6×His tag antibody. (D) Quantitative result of ELISA*, P < 0.05, compared with control antibody treatment.

Mentions: To purify scFv protein, cells were grown for an additional 12 or 32 h with IPTG induction at 16°C and then harvested by centrifugation. Protein purification was performed using Ni2+ affinity chromatography, and the purified proteins were visualized by SDS-PAGE using 12% (v/v) polyacrylamide gels. The concentrations of purified proteins from four different constructs are 0.13, 0.29, 0.115, and 0.38 mg/mL, respectively. After SDS-PAGE analysis, the target protein was further transferred onto a PVDF membrane, and anti-6×His tag antibody was used to confirm the presence of the expressed scFv protein. Protein purification and western blotting results showed that all four different fusion proteins were purified successfully (Figure 4A), and that all four could be recognized by the anti-6×His tag antibody during western blotting (Figure 4B).


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)

Protein purification and identification. (A) Protein purification. The purified protein was determined by SDS-PAGE. M: Molecular weight of marker proteins; Lane 1–4: the purified proteins for pACYC-Duet-scFv, pACYC-Duet-scFv-skp, pET28a-scFv, and pET32a-scFv, respectively. (B) Western blotting results. Lane 1–4: the detected bands for pACYC-Duet-scFv, pACYC-Duet-scFv-skp, pET28a-scFv, and pET32a-scFv, respectively. (C) ELISA. TLH antigen were coated on 96-well plates in triplicate (5 μ g/mL, 100 μ L/well), and the purified scFv proteins were added to the reaction wells after blocking and washing. The binding activities of the four purified proteins were determined using an anti-6×His tag antibody. (D) Quantitative result of ELISA*, P < 0.05, compared with control antibody treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Protein purification and identification. (A) Protein purification. The purified protein was determined by SDS-PAGE. M: Molecular weight of marker proteins; Lane 1–4: the purified proteins for pACYC-Duet-scFv, pACYC-Duet-scFv-skp, pET28a-scFv, and pET32a-scFv, respectively. (B) Western blotting results. Lane 1–4: the detected bands for pACYC-Duet-scFv, pACYC-Duet-scFv-skp, pET28a-scFv, and pET32a-scFv, respectively. (C) ELISA. TLH antigen were coated on 96-well plates in triplicate (5 μ g/mL, 100 μ L/well), and the purified scFv proteins were added to the reaction wells after blocking and washing. The binding activities of the four purified proteins were determined using an anti-6×His tag antibody. (D) Quantitative result of ELISA*, P < 0.05, compared with control antibody treatment.
Mentions: To purify scFv protein, cells were grown for an additional 12 or 32 h with IPTG induction at 16°C and then harvested by centrifugation. Protein purification was performed using Ni2+ affinity chromatography, and the purified proteins were visualized by SDS-PAGE using 12% (v/v) polyacrylamide gels. The concentrations of purified proteins from four different constructs are 0.13, 0.29, 0.115, and 0.38 mg/mL, respectively. After SDS-PAGE analysis, the target protein was further transferred onto a PVDF membrane, and anti-6×His tag antibody was used to confirm the presence of the expressed scFv protein. Protein purification and western blotting results showed that all four different fusion proteins were purified successfully (Figure 4A), and that all four could be recognized by the anti-6×His tag antibody during western blotting (Figure 4B).

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
Related in: MedlinePlus