Expression and purification of biologically active human granulocyte-macrophage colony stimulating factor (hGM-CSF) using silkworm-baculovirus expression vector system

https://doi.org/10.1016/j.pep.2019.03.010Get rights and content

Highlights

  • Recombinant human GM-CSF was highly purified from silkworm-BEVS.

  • The rhGM-CSF exhibited comparable activities to that of commercial GM-CSF.

  • Active rhGM-CSF could be commercially produced at lower cost.

Abstract

Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a hematopoietic growth factor. It is widely employed as a therapeutic agent targeting neutropenia in cancer patients undergoing chemotherapy and in patients with AIDS or after bone marrow transplantation. In this study, we constructed the recombinant baculoviruses for the expression of recombinant hGM-CSF (rhGM-CSF) with two small affinity tags (His-tag and Strep-tag) at the N or C-terminus. Compared to N-tagged rhGM-CSF, C-tagged rhGM-CSF was highly recovered from silkworm hemolymph. The purified rhGM-CSF proteins migrated as a diffuse band and were confirmed to hold N-glycosylations. A comparable activity was achieved when commercial hGM-CSF was tested as a control. Considering the high price of hGM-CSF in the market, our results and strategies using silkworm-baculovirus system can become a great reference for mass production of the active rhGM-CSF at a lower cost.

Introduction

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was first identified as a cytokine in a mouse lung tissue-conditioned medium [1]. It contributes to the stimulation of proliferation, differentiation, and survival of neutrophils, macrophages, and bone marrow-associated cells [2]. It also stimulates erythroid and megakaryocyte progenitor cells in synergy with other cytokines like stem cell factor (SCF) and interleukin 3 (IL-3). The production of GM-CSF could be found in many cell types, including stromal cells, macrophages, endothelial cells, fibroblasts and T cell [3], and its biological activities are executed when it binds with the GM-CSF receptor, which is thereafter phosphorylated by kinases from Janus kinase (JAK) family [4].

Human mature GM-CSF (hGM-CSF) is secreted as a 127-amino acid glycoprotein composed of a two-stranded antiparallel β-sheet and a four α-helix bundle [5]. It is an important therapeutic growth factor used in the treatment of neutropenia, myeloid leukemia, and aplastic anemia [6]. Furthermore, it is also found to prevent infections during bone marrow transplantation by accelerating neutrophil formation [7] and recognized as a critical cytokine in the development and progression of inflammatory arthritis [8]. Because of these clinical applications and the significant increase in demand, many attempts have been made to produce the biologically active recombinant hGM-CSF (rhGM-CSF) in large scale. However, the yield of purified proteins was lower than expected due to the toxicity of the recombinant product. For instance, in E. coli, overexpression of rhGM-CSF leads to the formation of the inclusion body because of the misfolding [9,10]. Transfected mammalian cells were also not preferred for the mass production of hGM-CSF due to low expression levels, high cost, and the presence of contaminating CSFs secreted by the cultured mammalian cells themselves [11].

The baculovirus expression vector system (BEVS) is widely used to express eukaryotic recombinant proteins because it allows us to obtain the proteins with mammalian-like posttranslational modifications [12]. In a previous study, a rhGM-CSF protein was expressed and purified from cultured insect cells [13]. However, to some extent, both the yield and the bioactivity seems not sufficient in the sense of mass production. Recently, our group has established the detailed protocols in silkworm (Bombyx mori) larvae-based BEVS and improved significantly on the expression and purification procedures to achieve recombinant proteins in both satisfactory quality and considerable quantity [14,15]. We revealed that silkworm-BEVS has a higher production efficiency than the culture cell expression system [16]. In this study, we demonstrated that the silkworm-BEVS is efficient for the large-scale production of biologically active rhGM-CSF.

Section snippets

Cells and silkworm strains

The NIAS-Bm-Oyanagi2 (BmO2: kindly provided from Dr. Imanishi) was cultured in IPL-41 medium (Sigma, St. Louis, MO) with 10% fetal bovine serum (Gibco, Grand Island, NY) at 27 °C. The silkworm strain n17 used for protein production was supplied by the silkworm stock center of Kyushu University supported by the Japan National BioResource Project. Detailed information for this silkworm strain could be found at https://shigen.nig.ac.jp/silkwormbase/ViewStrainDetail.do?name=n17. The silkworm larvae

Construction and expression of rhGM-CSF

Human GM-CSF consists of 127 amino acid residues which contain a 16-amino acid (aa 1–16) residues of secretion signal peptide (SP). To secrete the protein into cell culture medium or silkworm hemolymph efficiently, an endogenous signal peptide of silkworm 30K protein [19] was inserted into the N-terminus of hGM-CSF cDNA without the native SP of hGM-CSF. For facilitating the protein purification, Histidine 8 (H8)-tag, Strep-tag and Histidine 6 (H6)-tag were added to either the N- or C-terminal

Conclusions

In this study, rhGM-CSF was successfully purified by using silkworm-BEVS through silkworm larvae. We constructed two forms rhGM-CSF fused with the His-Strep tandem tag at N- or C-terminal. Both of the constructs were secreted successfully into the silkworm hemolymph, but not sufficiently into the cell culture media using cultured silkworm cells. The rhGM-CSFs were purified by a two-step purification. 1.1 mg of N-tagged rhGM-CSF and 3.8 mg of C-tagged rhGM-CSF were obtained from 1 ml of the

Conflict of interest

The authors declare no conflict of interest.

Contributor statement

YK made substantial contribution on this work and perform most of the experiments with the help of the other listed co-authors. JX, TK and JML involved in study concept and design, supervised this work, and validated experimental data. All authors made critical revisions of the manuscript and approved the final manuscript.

Acknowledgments

We thank Dr. Imanishi (National Institute of Agrobiological Sciences, Japan) for kindly providing the NIAS-Bm-oyanagi2 (BmO2) cell line. This work was supported by the Japan Science and Technology Agency (JST) for the Program for Creating Start-ups from Advanced Research and Technology (START Program).

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