F Rezaie1, F Davami1, K Mansouri2, S Agha Amiri1, R Fazel1, R Mahdian3, N Davoudi1, S Enayati1, M Azizi1, V Khalaj1. 1. Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran. 2. Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran. 3. Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Abstract
AIMS: The Escherichia coli expression system is highly effective in producing recombinant proteins. However, there are some limitations in this system, especially in obtaining correctly folded forms of some complex proteins such as Fab fragments. To improve the solubility and folding quality of Fab fragments, we have examined the effect of simultaneous application of a SUMO fusion tag, EnBase® cultivation mode and a redox mutant strain in the E. coli expression system. METHODS AND RESULTS: A bicistronic gene construct was designed to express an antivascular endothelial growth factor (VEGF) Fab fragment as a model system. The construct contained a dual SUMO fusion gene fragment to encode SUMO-tagged heavy and light chains. While the expression of the construct in batch cultures of BL21 or SHuffle® transformants produced insoluble and unfolded products, the induction of the transformants in EnBase® medium resulted in soluble and correctly folded Fab fragment, reaching as high as 19% of the total protein in shuffle strain. The functional assays indicated that the biological activity of the target Fab is similar to the commercial anti-VEGF, Lucentis® . CONCLUSIONS: This study demonstrated that the combination of SUMO fusion technology, EnBase® cultivation system and recruiting a redox mutant of E. coli can efficiently enhance the solubility and productivity of recombinant Fab fragments. SIGNIFICANCE AND THE IMPACT OF THE STUDY: The presented strategy provides not only a novel method to produce soluble and active form of an anti-VEGF Fab but also may use in the efficient production of other antibody fragments.
AIMS: The Escherichia coli expression system is highly effective in producing recombinant proteins. However, there are some limitations in this system, especially in obtaining correctly folded forms of some complex proteins such as Fab fragments. To improve the solubility and folding quality of Fab fragments, we have examined the effect of simultaneous application of a SUMO fusion tag, EnBase® cultivation mode and a redox mutant strain in the E. coli expression system. METHODS AND RESULTS: A bicistronic gene construct was designed to express an antivascular endothelial growth factor (VEGF) Fab fragment as a model system. The construct contained a dual SUMO fusion gene fragment to encode SUMO-tagged heavy and light chains. While the expression of the construct in batch cultures of BL21 or SHuffle® transformants produced insoluble and unfolded products, the induction of the transformants in EnBase® medium resulted in soluble and correctly folded Fab fragment, reaching as high as 19% of the total protein in shuffle strain. The functional assays indicated that the biological activity of the target Fab is similar to the commercial anti-VEGF, Lucentis® . CONCLUSIONS: This study demonstrated that the combination of SUMO fusion technology, EnBase® cultivation system and recruiting a redox mutant of E. coli can efficiently enhance the solubility and productivity of recombinant Fab fragments. SIGNIFICANCE AND THE IMPACT OF THE STUDY: The presented strategy provides not only a novel method to produce soluble and active form of an anti-VEGF Fab but also may use in the efficient production of other antibody fragments.