Expression of human prolactin in HEK293T using different transfection reagents
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2017
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ANNUAL MEETING OF THE BRAZILIAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, 46th
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INTRODUCTION
Prolactin is a hormone produced by the pituitary gland with numerous functions, such as lactation, reproduction, osmotic and
immune regulation. This hormone is upregulated in cases of lack of lactation, infertility and cancer. Recombinant prolactin has
been produced in Escherichia coli with an initial methionine which may cause immunological reactions, or in its authentic form
in mammalian cells. Our laboratory has already synthesized human prolactin (hPRL) without initial methionine in E. coli
periplasm and Chinese hamster ovary cells. CHO cells have been widely used in the synthesis of human recombinant proteins
because of their similarity with human post-translational modifications as glycosylation. The HEK293, a human embryonic
kidney cell, can do diverse glycosylation depend on culture conditions.
OBJECTIVES
This work compares different transfection reagents in the production of hPRL in HEK293T.
MATERIALS AND METHODS
The hPRL cDNA was inserted into the pEDdc vector donated by the Genetics Institute, USA. Three transfection reagents were
used: LipofectamineTM (Thermo), XfectTM (Clontech), and ExpiFectamineTM (Thermo). HEK293T cells, a human strain, were
cultured in 10 cm² Ø petri dishes with RPMI 1640 medium with 10% fetal bovine serum (FBS). After transfection, the medium
was changed to serum free CHO-S-SFM II (Invitrogen, USA). 100% of the medium was collected and changed every two days.
The collected medium was stored at -80°C. Samples were analyzed by SDS-PAGE, Western blotting and HPLC.
DISCUSSION AND RESULTS
The glycosylated and non-glycosylated hPRL forms secreted into the culture medium were confirmed by Western blot and RPHPLC
in the three transfected cultures in recombinant human cells. The reagent with the best result was Xfect (2 μg/mL),
followed by Lipofectamine (1.6 μg/mL) and Expifectamine (1.2 μg/mL).
CONCLUSION
The transient expression of hPRL using HEK293T cells enable laboratory production of glycosylated hPRL for future studies of
N-glycans produced by these cells.
Como referenciar
SILVA, FELIPE D.; SUZUKI, MIRIAM; OLIVEIRA, JOAO; FREIRE, RENAN; BARTOLINI, PAOLO; SOARES, CARLOS; AFFONSO, REGINA. Expression of human prolactin in HEK293T using different transfection reagents. In: ANNUAL MEETING OF THE BRAZILIAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, 46th, July 27-30, 2017, Águas de Lindóia, SP. Abstract... São Paulo, SP: Sociedade Brasileira de Bioquímica e Biologia Molecular, 2017. Disponível em: http://repositorio.ipen.br/handle/123456789/28436. Acesso em: 19 Apr 2024.
Esta referência é gerada automaticamente de acordo com as normas do estilo IPEN/SP (ABNT NBR 6023) e recomenda-se uma verificação final e ajustes caso necessário.