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| A Vector System for the Production of Single-chain Fv-Fc Fusions in Pichia pastoris |
| WANG Ding-ding1, SU Man-man2, HU Li-li1, YUAN Li-ying4, SUN Yan1, WANG Ju3, YAN Wei-qun2 |
1. Institute of Life Science and Biological Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China;
2. College of Pharmaceutical, Jilin University, Changchun 130021, China;
3. College of Life Science and Technology, Jinan University, Guangzhou 510632, China;
4. The 3rd Kindergarten Affiliated to Jilin University, Changchun 130021, China |
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Abstract Recombinant antibodies, especially ScFv fragments, can be applied as detection reagents and even substitute for some reagents used in immunoassays such as antibody-enzyme conjugates. For ScFv fragments, a universal system available is necessary. A vector system was constructed based on pPICZα/Fc, in which the hinge, CH2 and CH3 domains (Fc fragment) of human IgG1 and His-tag were cloned into the Pichia expression vector pPICZα. Two fragments of ScFv were introduced into pPICZα/Fc, which can bind HBsAg and rabies virus antigen, to yield the expression cassette pPICZα/ScFv-Fc. Following fermentation in a 1-liter reactor, the fusions were expressed at high levels in the methylotrophic yeast Pichia pastoris, secreted as dimeric forms in the culture, and purified by protein A column chromatography. The expression yield can reach 20~30mg/L of culture medium. The ScFv-Fc fusion proteins retain the biological binding ability of the parent ScFv. Furthermore, the successful expression and maintenance of the binding activity verify the efficacy of the vector system for use as detection reagents in vitro, by reacting with the specific antigens and being readily detected using general anti-human IgG antibodies.
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Received: 10 June 2011
Published: 25 August 2011
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