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.

Integrated process development: The key to improve Fab production in E. coli

“…Furthermore, these proteins can overload host cell folding capacities or be exposed to proteolysis by host proteases. [ 18,19 ] Low expression yields additionally challenge the scale‐up and DSP, significantly affecting production costs. [ 20 ]…”

“…Furthermore, these proteins can overload host cell folding capacities or be exposed to proteolysis by host proteases. [18,19] Low expression yields additionally challenge the scale-up and DSP, significantly affecting production costs. [20] Examples of such challenging proteins are antibody fragments, such as antigen binding fragments (Fabs) or single-chain variable fragments (scFvs), which can be produced in E. coli.…”

Predictive Approaches to Guide the Expression of Recombinant Vaccine Targets in Escherichia Coli: A Case Study Presentation Utilising Absynth Biologics Ltd Proprietary C.Difficile Vaccine Antigens

“…Different groups of chaperones exist in E.coli to aid protein folding, prevent aggregation and/or degradation and co-expression of these factors have shown to increase protein yields and solubility of certain recombinant targets [14,15]. Great efforts have been focused on optimisation of the culture conditions such as the growth medium, inducer concentrations [16,17], induction temperature [18,19] and the isolation, re-solubilisation and puri cation of proteins from inclusion bodies [4,7,8]. Other strategies employed include the targeting of proteins to different cellular compartments, for example the periplasmic space where an oxidising environment and lower protease concentration has been shown to increase protein production and stability [20].…”

Predictive approaches to guide the expression of recombinant vaccine targets in Escherichia coli: a case study presentation utilising Absynth Biologics Ltd. proprietary Clostridium difficile vaccine antigens

“…Different groups of chaperones exist in E. coli to aid protein folding and prevent aggregation and/or degradation, and co-expression of these factors have shown to increase protein yields and solubility of certain recombinant targets (De Marco et al 2007 ; Gupta and Shukla 2016 ; Nishihara et al 1998 ). Great efforts have been focused on optimisation of the culture conditions such as the growth medium, inducer concentrations (Hemmerich et al 2017 ; Ramirez et al 1994 ), induction temperature (Barazesh et al 2019 ; Schein and Noteborn 1988 ) and the isolation, re-solubilisation and purification of proteins from inclusion bodies (Kaur and Kumar 2017 ; Oberg et al 1994 ; Przybycien et al 1994 ). Other strategies employed include the targeting of proteins to different cellular compartments, for example, the periplasmic space where an oxidising environment and lower protease concentration has been shown to increase protein production and stability (Malik 2016 ).…”