Two Simple Methods for Optimizing the Production of "Difficult-to-Express" GnRH-DFF40 Chimeric Protein

Abstract

Purpose: GnRH-DFF40 (gonadotropin releasing hormone - DNA fragmentation factor 40) is a humanized recombinant immunotoxin and serves as a prospective candidate for targeted therapy of gonadotropin releasing hormone receptor (GnRHR) overexpressing malignancies. However, its production in Escherichia coli in a soluble and functional form still remains a challenge. Here we introduce two successful and reproducible conditions for production and purification of "difficult-to-express" GnRH-DFF40 protein. Methods: A synthetic codon optimized GnRH-DFF40 fusion gene was cloned in pET28a plasmid. Two methods including high cell density IPTG induction (HCDI) and autoinduction method (AIM) with a focus on obtaining high cell density have been investigated to enhance the protein production in (E. coli). Moreover, to obtain higher protein production several factors in the AIM method including carbon sources, incubation time and temperature, plasmid stability and double colony selection, were optimized. Results: Remarkable amounts of soluble GnRH-DFF40 protein were achieved by both methods. Cell density and protein yields in AIM was about 1.5 fold higher than that what obtained using HCDI. Initial screening showed that 25ºC is better to achieve higher protein production in both methods. pH alterations in AIM were maintained in a more constant level at 25ºC and 37ºC temperatures without any detrimental effects on cell growth during protein production phase up to 21 hours after incubation. Plasmid stability during growth and expression induction phase was maintained at a high level of 98% and 96% for AIM and HCDI methods, respectively. After parameter optimization and double colony selection in AIM, a very high yield of recombinant protein was achieved (528.3 mg/L). Conclusion: With the optimization of these high cell density expression methods, reproducible manifold enhancement of soluble protein yields can be achieved for "difficult-to-express" GnRH-DFF40 compared to conventional expression methods.