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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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SDS-PAGE analysis. (A) SDS-PAGE analysis of expressed TLH antigen protein. Lane 1: Control (empty vector pGEPi, IPTG induced); Lane 2: pGEPi-tlh expressed product (IPTG induced); Lane 3, 4: Extracted TLH protein (without 6×His tag); (B) Expression of different scFv constructs in E. coli BL21. M: Molecular weight of marker proteins; Lanes 1, 4, and 6: negative control (pACYC-Duet, pET28a, and pET32a, respectively); Lane 2, 3, 5, and 7: the expressed products of pACYC-Duet-scFv, pACYC-Duet-scFv-skp, pET28a-scFv, and pET32a-scFv, respectively.
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Figure 2: SDS-PAGE analysis. (A) SDS-PAGE analysis of expressed TLH antigen protein. Lane 1: Control (empty vector pGEPi, IPTG induced); Lane 2: pGEPi-tlh expressed product (IPTG induced); Lane 3, 4: Extracted TLH protein (without 6×His tag); (B) Expression of different scFv constructs in E. coli BL21. M: Molecular weight of marker proteins; Lanes 1, 4, and 6: negative control (pACYC-Duet, pET28a, and pET32a, respectively); Lane 2, 3, 5, and 7: the expressed products of pACYC-Duet-scFv, pACYC-Duet-scFv-skp, pET28a-scFv, and pET32a-scFv, respectively.

Mentions: After the TLH protein was expressed, the total protein was analyzed by SDS-PAGE, and the gel was stained using 0.3 M CuCl2, and visualized against a black background. The target TLH protein bands were cut and mashed with PBS buffer. After harvested by centrifugation, the extracted supernatant containing the TLH protein was visualized by SDS-PAGE. As shown in Figure 2A, the TLH antigen protein was expressed successfully (Figure 2A, lane 2), and the target TLH protein was extracted with a high purity (Figure 2A, lane 3, 4). The expressed TLH protein contains HA and Myc tags (without 6×His tag), and was used as the immobilized antigen during ELISA.


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)

SDS-PAGE analysis. (A) SDS-PAGE analysis of expressed TLH antigen protein. Lane 1: Control (empty vector pGEPi, IPTG induced); Lane 2: pGEPi-tlh expressed product (IPTG induced); Lane 3, 4: Extracted TLH protein (without 6×His tag); (B) Expression of different scFv constructs in E. coli BL21. M: Molecular weight of marker proteins; Lanes 1, 4, and 6: negative control (pACYC-Duet, pET28a, and pET32a, respectively); Lane 2, 3, 5, and 7: the expressed products of pACYC-Duet-scFv, pACYC-Duet-scFv-skp, pET28a-scFv, and pET32a-scFv, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: SDS-PAGE analysis. (A) SDS-PAGE analysis of expressed TLH antigen protein. Lane 1: Control (empty vector pGEPi, IPTG induced); Lane 2: pGEPi-tlh expressed product (IPTG induced); Lane 3, 4: Extracted TLH protein (without 6×His tag); (B) Expression of different scFv constructs in E. coli BL21. M: Molecular weight of marker proteins; Lanes 1, 4, and 6: negative control (pACYC-Duet, pET28a, and pET32a, respectively); Lane 2, 3, 5, and 7: the expressed products of pACYC-Duet-scFv, pACYC-Duet-scFv-skp, pET28a-scFv, and pET32a-scFv, respectively.
Mentions: After the TLH protein was expressed, the total protein was analyzed by SDS-PAGE, and the gel was stained using 0.3 M CuCl2, and visualized against a black background. The target TLH protein bands were cut and mashed with PBS buffer. After harvested by centrifugation, the extracted supernatant containing the TLH protein was visualized by SDS-PAGE. As shown in Figure 2A, the TLH antigen protein was expressed successfully (Figure 2A, lane 2), and the target TLH protein was extracted with a high purity (Figure 2A, lane 3, 4). The expressed TLH protein contains HA and Myc tags (without 6×His tag), and was used as the immobilized antigen during ELISA.

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