Expression of glycosylated human prolactin in HEK293 cells and related N-glycan composition analysis

Autor(es)

Silva, Felipe D. ^[1] ; Oliveira, Joao E. ^[1] ; Freire, Renan P. ^[1] ; Suzuki, Miriam F. ^[1] ; Soares, Carlos R. ^[1] ; Bartolini, Paolo ^[1]

Afiliação do(s) autor(es)

^[1] IPEN, CNEN SP, Biotechnol Ctr, Ave Prof Lineu Prestes 2242, BR-05508000 Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: AMB EXPRESS; v. 9, n. 1 AUG 29 2019.
• Citações Web of Science: 0

Resumo

Prolactin (PRL) is a hormone produced by the pituitary gland with innumerable functions, such as lactation, reproduction, osmotic and immune regulation. The present work describes the synthesis of hPRL in human embryonic kidney (HEK293) cells, transiently transfected with the pcDNA-3.4-TOPO (R) vector carrying the hPRL cDNA. A concentration of 20 mg/L, including glycosylated (G-hPRL) and non-glycosylated (NG-hPRL) human prolactin, was obtained, with 19% of G-hPRL, which is higher than that observed in CHO-derived hPRL ( 10%) and falling within the wide range of 5-30% reported for pituitary-derived hPRL. N-Glycoprofiling analysis of G-hPRL provided: (i) identification of each N-glycan structure and relative intensity; (ii) average N-glycan mass; (iii) molecular mass of the whole glycoprotein and relative carbohydrate mass fraction; (iv) mass fraction of each monosaccharide. The data obtained were compared to pituitary- and CHO-derived G-hPRL. The whole MM of HEK-derived G-hPRL, determined via MALDI-TOF-MS, was 25,123 Da, which is 0.88% higher than pit- and 0.61% higher than CHO-derived G-hPRL. The main difference with the latter was due to sialylation, which was sevenfold lower, but slightly higher than that observed in native G-hPRL. The ``in vitro{''} bioactivity of HEK-G-hPRL was fourfold lower than that of native G-hPRL, with which it had in common also the number of N-glycan structures. (AU)

• Processo FAPESP: 15/26058-0 - Estudo do perfil de N-glicosilação e dos sítios de ocupação em várias preparações de tireotropina humana (hTSH) hipofisária e recombinante
• Beneficiário: Carlos Roberto Jorge Soares
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 17/50332-0 - Capacitação científica, tecnológica e em infraestrutura em radiofármacos, radiações e empreendedorismo a serviço da saúde (PDIp)
• Beneficiário: Marcelo Linardi
• Modalidade de apoio: Auxílio à Pesquisa - Programa Modernização de Institutos Estaduais de Pesquisa