Enhancement of human erythropoietin production in Chinese hamster ovary cells through supplementation of 30Kc19-30Kc6 fusion protein

Highlights

• Improved soluble expression of 30Kc6 (expression partner) when expressed with 30Kc19 in E. coli.

• 30Kc19-30Kc6 fusion protein efficiently delivered cell-impermeable 30Kc6 cargo protein.

• Supplementation of 30Kc19-30Kc6 protein increased the viability and inhibited apoptosis.

• Mitochondrial membrane potential and ATP generation were increased in various cells.

• Specific EPO productivity was increased due to multi-functional properties of 30Kc19-30Kc6.

Abstract

The importance of anti-apoptosis in mammalian cell culture has been widely recognized. We reported previously that expression of Bombyx mori 30 K genes in Chinese hamster ovary (CHO) cells increases recombinant protein production by inhibiting apoptosis and enhancing specific productivity. However, previous studies have shown expression of the anti-apoptotic 30Kc6 protein as inclusion bodies in Escherichia coli. 30Kc19 protein, another silkworm hemolymph protein, has cell-penetrating and recombinant protein productivity-improving properties, and we found that it improves soluble expression of its partner. In this study, we fused 30Kc6 with 30Kc19 as an expression partner to express a soluble fused protein and to deliver the protein to cells. Supplementing the recombinant 30Kc19-30Kc6 fusion protein in cell culture medium increased viability, effectively penetrated the cells, and inhibited CHO cell apoptosis by 68%. Moreover, the mitochondrial membrane potential and ATP generation also increased by 50% and 33%, respectively. Erythropoietin (EPO) productivity increased by > 30% because of the anti-apoptotic effect and increased specific productivity. These results demonstrate the potential use of this fusion protein as a supplement in mammalian cell culture during production of biopharmaceutical proteins.

Introduction

The Chinese hamster ovary (CHO) cell line has been widely used as a mammalian host cell [1], [2] for producing recombinant glycoproteins, such as interferon-β, monoclonal antibodies, and erythropoietin (EPO) [3], [4], [5] because of its advantageous properties such as high production; suitability for large-scale culture; and similarities in the glycan structures produced from CHO cells to those of human glycoproteins [6], [7], [8]. However, there is a difficulty in maintaining high cell growth and specific productivity because cells are subjected to programmed cell death (PCD). This is a common problem encountered in cell culture; which decreases recombinant protein productivity. During the culture process, nutrient depletion, hypoxia, waste by-product accumulation, and other factors lead to cell apoptosis [9], [10], [11]. Various attempts have been performed to inhibit apoptosis, such as nutrient supplements [12], adding apoptosis inhibitors [13], [14], [15], [16], and expressing anti-apoptotic genes [17], [18]. Traditional methods, such as addition of sodium butyrate have been reported to increase recombinant protein production [19], [20], [21]. However, it also induces apoptosis by inhibiting histone deacetylation [22], [23], which reduces cell viability and productivity. Thus, despite the beneficial effect on protein expression; the use of sodium butyrate is compromised by its cytotoxic effect.

Silkworm hemolymph consists of a group of structurally related proteins with a molecular weight of approximately 30 kDa, including 30Kc6, 30Kc12, 30Kc19, 30Kc21, and 30Kc23. During the fifth instar larval to early pupal stages, these “30 K proteins” are synthesized in fat body cells and accumulated in the hemolymph [24], [25]. During metamorphosis from larva to pupa, these proteins are transferred from the hemolymph to fat body cells and are deposited there until use [26], [27]. We have demonstrated previously that silkworm hemolymph and 30 K proteins exhibit an anti-apoptotic effect in various cells by adding the protein to culture medium or by gene expression [18], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39]. 30 K proteins also enhance production of recombinant EPO, interferon-β, and monoclonal antibodies; increase glycosylation, cell growth, and viability in various cells; and have an enzyme-stabilizing effect [17], [40], [41], [42], [43], [44], [45], [46]. Additionally, the 30Kc19 protein has a cell-penetrating property when added to culture medium; thus, it can be applied to deliver cargo proteins, as it can penetrate cell membranes and stabilize cargo proteins [47], [48].

In this study, soluble expression and purification of a 30Kc19-30Kc6 fusion protein in Escherichia coli was used for inhibiting apoptosis and improving recombinant protein production in CHO cells. Considering that EPO is one of the most demanded recombinant proteins, we added the 30Kc19-30Kc6 fusion protein to CHO cell culture medium to increase productivity of recombinant human EPO (rHuEPO) in serum-free media. This 30Kc19-30Kc6 fusion protein has advantageous properties of both 30Kc19 and 30Kc6, which are soluble, cell-penetrating, anti-apoptotic, and productivity-enhancing properties. This work demonstrates the potential use of this multifunctional protein in mammalian cell culture for the production of recombinant proteins.