Production, purification and characterization of recombinant human R-spondin1 (RSPO1) protein stably expressed in human HEK293 cells

Autor(es)

Levin, Gabriel ^[1] ; Koga, Bruna Andrade Aguiar ^{[1, 2]} ; Belchior, Gustavo Gross ^[1] ; Carreira, Ana Claudia Oliveira ^{[1, 2]} ; Sogayar, Mari Cleide ^{[3, 1]}

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Cell & Mol Therapy Ctr NUCEL, Sch Med, Edificio NUCEL, Rua Pangare, 100 Cidade Univ, BR-05360130 Sao Paulo, SP - Brazil

^[2] Univ Sao Paulo, Sch Vet Med & Anim Sci, Dept Surg, BR-13635900 Sao Paulo, SP - Brazil

^[3] Univ Sao Paulo, Inst Chem, Dept Biochem, BR-05508000 Sao Paulo, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: BMC Biotechnology; v. 20, n. 1 JAN 20 2020.
• Citações Web of Science: 0

Resumo

Background The R-Spondin proteins comprise a family of secreted proteins, known for their important roles in cell proliferation, differentiation and death, by inducing the Wnt pathway. Several studies have demonstrated the importance of RSPOs in regulation of a number of tissue-specific processes, namely: bone formation, skeletal muscle tissue development, proliferation of pancreatic beta-cells and intestinal stem cells and even cancer. RSPO1 stands out among RSPOs molecules with respect to its potential therapeutic use, especially in the Regenerative Medicine field, due to its mitogenic activity in stem cells. Here, we generated a recombinant human RSPO1 (rhRSPO1) using the HEK293 cell line, obtaining a purified, characterized and biologically active protein product to be used in Cell Therapy. The hRSPO1 coding sequence was synthesized and subcloned into a mammalian cell expression vector. HEK293 cells were stably co-transfected with the recombinant expression vector containing the hRSPO1 coding sequence and a hygromycin resistance plasmid, selected for hygro(R) and subjected to cell clones isolation. Results rhRSPO1 was obtained, in the absence of serum, from culture supernatants of transfected HEK293 cells and purified using a novel purification strategy, involving two sequential chromatographic steps, namely: heparin affinity chromatography, followed by a molecular exclusion chromatography, designed to yield a high purity product. The purified protein was characterized by Western blotting, mass spectrometry and in vitro (C2C12 cells) and in vivo (BALB/c mice) biological activity assays, confirming the structural integrity and biological efficacy of this human cell expression system. Furthermore, rhRSPO1 glycosylation analysis allowed us to describe, for the first time, the glycan composition of this oligosaccharide chain, confirming the presence of an N-glycosylation in residue Asn137 of the polypeptide chain, as previously described. In addition, this analysis revealing the presence of glycan structures such as terminal sialic acid, N-acetylglucosamine and/or galactose. Conclusion Therefore, a stable platform for the production and purification of recombinant hRSPO1 from HEK293 cells was generated, leading to the production of a purified, fully characterized and biologically active protein product to be applied in Tissue Engineering. (AU)

• Processo FAPESP: 17/01072-6 - Análise do potencial terapêutico da proteína recombinante humana RSPO1 na regeneração de intestino delgado em modelo animal utilizando tecnologias de engenharia tecidual
• Beneficiário: Gabriel Levin
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Doutorado Direto
• Processo FAPESP: 15/11128-3 - Análise do potencial terapêutico da proteína recombinante humana RSPO1 na regeneração de intestino delgado em modelo animal utilizando tecnologias de Engenharia tecidual
• Beneficiário: Gabriel Levin
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto
• Processo FAPESP: 16/05311-2 - Medicina regenerativa visando à terapia de doenças crônico-degenerativas (câncer e diabetes)
• Beneficiário: Mari Cleide Sogayar
• Modalidade de apoio: Auxílio à Pesquisa - Temático