Controlled In Vivo Processing Of Scfv-Mbp Fusion Protein: Towards High Level Production Of Soluble And Functional Scfv- Based Biopharmaceutical In Bacterial System
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Date
2015-06
Authors
Aly Atef, Aly Shoun
Journal Title
Journal ISSN
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Publisher
Universiti Sains Malaysia
Abstract
In the past two decades, the bacterial expression of antibody fragments has shown great dependence in the rapid expansion and a key success in antibody engineering. Bacterial expression and antibody engineering provide a suitable means of creating antigen binding fragments for isolation, evaluation and production of antibody. In this study we succeeded to design specific primer for anti-HIV1-CA scFv, which was used in PCR amplification of anti-HIV1-CA scFv, MBP and vector pMXB10Hp24-6His. Subsequently, restriction enzyme analysis has been done using Nde1-R/Not1-F enzymes then the restricted insert and vector were ligated. The constructed plasmid, pMXB10-MBP-TEV-scFv46 was transferred into E.Coli DH5-α competent cells. Numerous positive clones were presented on the transformed plate and clones were randomly selected to be confirmed by colony PCR which showed the band for pMXB10-MBP-TEV-scFv46 clearly. The plasmid DNA was harvested, purified then transformed into NiCo21 (DE3), SHuffle (C3029) as competent cells for protein expression. The colony PCR was performed in order to confirm the presence of pMXB10-MBP-TEV-scFv46 clones in the transformant. Starter cultures were prepared in order to optimize expression of the protein of interest; (NiCo21-TEVEco and SHuffle-TEVEco) competent cells were used. Indeed, the expression of MBP-scFv-46 using NiCo21-TEVEco as competent cell was mainly present in the insoluble fraction. Immunoblot analysis with the anti-6His antibody revealed that none of MBP-scFv-46 proteins was present in the soluble fraction. However, Almost 90%-95% of MBP-scFv fusion protein has been cleaved by TEV protease using SHuffle-TEVEco. Immunoblot analysis also showed that almost 40% of scFv was present in the soluble fraction, which mean that we succeeded to develop novel system in order to achieve Controlled in vivo processing of MBP-scFv fusion proteins: Towards high level production of soluble scFv from E.Coli.
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Keywords
Antibody engineering