Efficient Interleukin-21 Production by Optimization of Codon and Signal Peptide in Chinese Hamster Ovarian Cells

Abstract

Interleukin-21 is a common γ-chain cytokine that controls the immune responses of B cells, T cells, and natural killer cells. Targeting IL-21 to strengthen the immune system is promising for the development of vaccines as well as anti-infection and anti-tumor therapies. However, the practical application of IL-21 is limited by the high production cost. In this study, we improved IL-21 production by codon optimization and selection of appropriate signal peptide in CHO-K1 cells. Codon-optimized or non-optimized human IL-21 was stably transfected into CHO-K1 cells. IL-21 expression was 10-fold higher for codon-optimized than non-optimized IL-21. We fused five different signal peptides to codon-optimized mature IL-21 and evaluated their effect on IL-21 production. The best result (a 3-fold increase) was obtained using a signal peptide derived from human azurocidin. Furthermore, codon-optimized IL-21 containing the azurocidin signal peptide promoted IFN-γ secretion and STAT3 phosphorylation in NK-92 cells similar to codon-optimized IL-21 containing original signal peptide. Collectively, these results indicate that codon optimization and azurocidin signal peptides provide an efficient approach for the high-level production of IL-21 as a biopharmaceutical.

Evaluation of artificial signal peptides for secretion of two lysosomal enzymes in CHO cells

“…Signal peptides play a significant role in recombinant protein production. Several studies have suggested that using artificial signal peptides can improve the expression and secretion of a recombinant protein (19)(20)(21)(22)(23)(24). Importantly, altering signal peptide sequences is designed to change binding to the signal recognition particle, which ideally should not affect the characteristics of a recombinant protein.…”

“…These included antibody signal peptides from Ig heavy chain signal peptide 7 and Igκ light chain signal peptide 1, that exhibit the best secretion for 4 antibodies out of the 5 in CHO K1 cells, including Avastin, Remicade, Rituxan and Humira (19), and also from murine Igκ light chain, that shows 1.5-fold as effective as the expression of recombinant human coagulation factor VII using native signal peptide in CHO K1 cells (20). We also selected signal peptides derived from human albumin, that increases antibody production in CHO K1 cells by 50-60% compared to control signal peptide (21), and from human azurocidin 1, that increases recombinant interleukin-21 production in CHO K1 cell media by 7-fold compared to native signal peptide (22). In addition, we selected signal peptides from human serine protease 2 and human chymotrypsinogen B1, that proved to be 14.2-fold and 11.8-fold as effective as that of human albumin for recombinant Gaussia luciferase production in CHO cells (23).…”

Cell factory engineering: Challenges and opportunities for synthetic biology applications

“…Signal peptides have been engineered to improve the affinity to the SRP, which correlates with the translocation efficiency and yield of the recombinant protein. The azurocidin signal peptide was found to increase IL-21 secretion approximately 2.5-fold and the Ig κ signal peptide to enhance the expression of recombinant coagulation factor VII 1.5fold in CHO cells (Cho et al, 2019;L. Peng et al, 2016).…”

“…Signal peptides have been engineered to improve the affinity to the SRP, which correlates with the translocation efficiency and yield of the recombinant protein. The azurocidin signal peptide was found to increase IL‐21 secretion approximately 2.5‐fold and the Ig $$\kappa $$ signal peptide to enhance the expression of recombinant coagulation factor VII 1.5‐fold in CHO cells (Cho et al, 2019; L. Peng et al, 2016). Similarly, optimization of the signal peptide mediating secretion of antibodies in CHO cells resulted in an increase in the yields of anti‐HER2 antibody (Herceptin, 2.2‐fold), anti‐CD20 antibody (Rituxan, 2‐fold), and anti‐VEGF‐A antibody (Avastin, 3‐fold) (Haryadi et al, 2015; You et al, 2018).…”

Razor: annotation of signal peptides from toxins

“…In addition, understanding the presence or absence of SPs in the genes of interest is critical for choosing the appropriate recombinant protein expression and purification systems, as the intracellular accumulation of secretory proteins and toxins may be toxic to the host cells. Indeed, the ability of SPs to translocate proteins has been utilised in recombinant protein expression systems for high quality and quantity results (Futatsumori-Sugai and Tsumoto, 2010; Cho et al ., 2019; Karyolaimos et al ., 2019; Peng et al ., 2019).…”