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

GnRH-DFF40 (gonadotropin releasing hormone - DNA fragmentation factor 40) isa humanized recombinant immunotoxin and serves as a prospective candidate for targetedtherapy of gonadotropin releasing hormone receptor (GnRHR) overexpressing malignancies.However, its production in Escherichia coli in a soluble and functional form still remains achallenge. Here we introduce two successful and reproducible conditions for production andpurification of "difficult-to-express" GnRH-DFF40 protein. A synthetic codon optimized GnRH-DFF40 fusion gene was cloned in pET28aplasmid. Two methods including high cell density IPTG induction (HCDI) and autoinductionmethod (AIM) with a focus on obtaining high cell density have been investigated to enhance theprotein production in (E. coli). Moreover, to obtain higher protein production several factors inthe AIM method including carbon sources, incubation time and temperature, plasmid stabilityand double colony selection, were optimized. 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 usingHCDI. Initial screening showed that 25ºC is better to achieve higher protein production in bothmethods. pH alterations in AIM were maintained in a more constant level at 25ºC and 37ºCtemperatures without any detrimental effects on cell growth during protein production phaseup to 21 hours after incubation. Plasmid stability during growth and expression induction phasewas maintained at a high level of 98% and 96% for AIM and HCDI methods, respectively. Afterparameter optimization and double colony selection in AIM, a very high yield of recombinantprotein was achieved (528.3 mg/L). With the optimization of these high cell density expression methods, reproduciblemanifold enhancement of soluble protein yields can be achieved for "difficult-to-express"GnRH-DFF40 compared to conventional expression methods.