Efficient production of recombinant IL-21 proteins for pre-clinical studies by a two-step dilution refolding method

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Introduction

Interleukin 21 (IL-21) is a recently discovered type I cytokine and classified in the family, which includes IL-2, IL-4, IL-7, IL-9, IL-13 andIL-15, all share common gamma chain subunit in their receptors[1]. The production of IL-21 is limited to CD4+T and natural killer T (NKT)cells but the expression of its receptor has been detected on multiple cellular components of the innate and adaptive immune system as well as certain non-hematopoietic cells such as endothelial cells[2].IL-21induces B cell proliferation and differentiation[1] and maintains the germinal center response for antibody affinity maturation[3,4].IL-21en-hances CD4+T cell proliferation and regulates the development and maintenance of IL-17 producing helper T (Th17)[5,6] and follicular helper T (Tfh) cells[7,8]. IL-21 acts synergistically with IL-15 to promote the proliferation and cytotoxicity of CD8+Tcells[9], and prevents CD8+Tcell exhaustion during chronic infection[10–12]. IL-21 enhances the maturation and promotes the cytolytic function of nature killer (NK)cells [2,13].The pleiotropic immunostimulatory functions suggest that IL-21function may be used, or manipulated, for vaccinations, anti-tumor andanti-infection therapies. Indeed, in reported phase II clinical trials on renal cell carcinoma and melanoma, recombinant human IL-21 demon-strated an acceptable safety profile and encouraging activity in its useas a single agent[14]. Though promising, investigating the therapeutic applications of recombinant IL-21in vivoin pre-clinical animal modelsis limited by relatively high cost of commercial resource of recombinantIL-21. For example, 1 mg recombinant human or mouse IL-21 from Pepprotech (Rocky Hill, NJ, USA) will cost 5200 US dollars, which makesit more desirable to develop an efficient method to produce recombinantIL-21 for pre-clinicalin vivostudies.Recombinant IL-21 proteins expressed inEscherichia coli(E.coli)showsimilar activities with those produced from mammalian systems[15] andhave been used in clinical trials[14].Herewedescribeanoptimizedmethod for rapid and cost-effective production of recombinant IL-21usingE.coli. Compared to previously reported methods[15–17] ,thestep to refold denatured IL-21 proteins solubilized from inclusion bodies(IBs) is modified by using a sequential dilution and dialysis procedure,which helps to achieve high yield, cut down the running time and reducethe cost of reagents. In addition, a simple and efficient strategy to removeendotoxin is incorporated in the method, rendering the feasibility of thefinal products to be directly used forin vitroandin vivostudies.

Reference: Z. Chen et al. Efficient production of recombinant IL-21 proteins for pre-clinical studies by a two-step dilution refolding method Int Immunopharmacol (2013), http://dx.doi.org/10.1016/j.intimp.2013.02.017

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