Efficient Production of Recombinant Human (Pro)renin Utilizing a Decahistidine Tag Attached at the C-Terminus

Nakagawa, Tsutomu; Nishiuchi, Kazuhiro; Akaki, Jyunji; Iwata, H; Satou, Ryousuke; Suzuki, Fujio; Nakamura, Yukio

doi:10.1271/bbb.60455

Cited by 15 publications

(14 citation statements)

References 19 publications

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“…Each eluate containing renin was dialyzed against 10 mM phosphate buffer (pH 7.0). The purified renins showed a 40-43 kDa triplex band by silver staining after SDS-polyacrylamide gel electrophoresis (data not shown), as reported previously, 14) indicating no contamination by other proteins.…”

Section: Methodssupporting

confidence: 88%

“…A stable cell line producing H13Y mutant sheep angiotensinogen was established as described previously. 14) Briefly, expression plasmid pcDNA3-sAngn-H13Y was cotransfected with a plasmid, pmDHFR, into Chinese hamster ovary (CHO) cells deficient in the dihydrofolate reductase gene. G418-resistant clonal cells were treated with methotrexate (Sigma-Aldrich, St. Louis, MO) to enhance production of the recombinant protein.…”

Section: Methodsmentioning

confidence: 99%

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The Coexistence of Ser84 in Renin and His13 in Angiotensinogen Brings a pH Profile of Two Separate Peaks to the Reaction of Human Renin and Sheep Angiotensinogen

Iwata

Nakagawa

Yoshioka

et al. 2008

Bioscience, Biotechnology, and Biochemistry

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Section: Methodssupporting

confidence: 88%

Section: Methodsmentioning

confidence: 99%

The Coexistence of Ser84 in Renin and His13 in Angiotensinogen Brings a pH Profile of Two Separate Peaks to the Reaction of Human Renin and Sheep Angiotensinogen

Iwata

Nakagawa

Yoshioka

et al. 2008

Bioscience, Biotechnology, and Biochemistry

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“…The ability of recombinant (P)RR to bind its ligands, renin and prorenin, has been evaluated previously in real time using surface plasmon resonance in a BIAcore assay system. 16,19 Chinese hamster ovary cells harboring complementary DNA coding for human prorenin (383 amino-acid residues with a decahistidine at the C terminus) were maintained in conditioned Dulbecco's modified Eagle's medium according to the method described by Nakagawa et al 21 SDS-polyacrylamide gel electrophoresis and western blot analysis were used to determine the molecular size of prorenin. The secreted crude preparation of prorenin in the culture medium was purified as described above.…”

Section: Synthesis and Purification Of Recombinant (P)rr And Proreninmentioning

confidence: 99%

“…The secreted crude preparation of prorenin in the culture medium was purified as described above. 21 Real-time binding of prorenin to s(P)RR secreted by cultured HUVECs…”

Section: Synthesis and Purification Of Recombinant (P)rr And Proreninmentioning

confidence: 99%

Qualitative and quantitative analyses of (pro)renin receptor in the medium of cultured human umbilical vein endothelial cells

et al. 2011

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A 30-kDa protein in the medium of cultured human umbilical vein endothelial cells (HUVECs) was identified as (pro)renin receptor, (P)RR, by western blot analysis using anti-human (P)RR antibodies. The protein bound recombinant human prorenin with a K D of 4.0 nmol l À1 and activated prorenin. These observations suggest the presence of soluble (P)RR, s(P)RR, in the medium of cultured HUVECs. For quantification of the s(P)RR in the medium, an enzyme-linked immunosorbent assay (ELISA) was established. The quantitative range of the ELISA was validated over a nominal range of 7.5-300 pmol l À1 in the wells of a microtiter plate. The assay system showed good linearity (r 2 ¼0.99) with interassay (5.8-9.7%) and intraassay (2.1-7.0%) precision. Using this method, the concentration of s(P)RR in the culture medium of HUVECs was measured to be 32 pmol l À1 . Therefore, these results show qualitative and quantitative evidence that prorenin can be activated after binding to s(P)RR secreted from cultured HUVECs.

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“…3 A adição da cauda de histidina em proteínas recombinantes tem sido altamente efetiva, proporcionando a purificação da proteína alvo em uma única etapa com alto teor de pureza. 26 Como exemplos de proteínas recombinantes com cauda de histidina purificadas por IMAC em nível de bancada, podem-se citar pró-renina humana, 83 β-galactosidase e peroxidase de raiz forte 84,85 e, em nível industrial, a pró-insulina humana. 86 Pró-renina humana produzida em células de ovário de hamster chinês (CHO), tendo uma cauda de 10 histidinas, foi purificada por IMAC, fazendo-se uso de colunas comerciais Talon ® -Co 2+ .…”

Section: Exemplos De Aplicações De Imacunclassified

Cromatografia de afinidade por íons metálicos imobilizados (IMAC) de biomoléculas: aspectos fundamentais e aplicações tecnológicas

Bresolin¹,

Miranda²,

Bueno³

2009

Quím. Nova

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Recebido em 16/7/08; aceito em 16/12/08; publicado na web em 11/5/09 IMMOBILIZED METAL-ION AFFINITY CHROMATOGRAPHY (IMAC) OF BIOMOLECULES: FUNDAMENTAL ASPECTS AND TECHNOLOGICAL APPLICATIONS. Immobilized Metal Ion Affinity Cromatography -IMAC -is a group-specific based adsorption applied to the purification and structure-function studies of proteins and nucleic acids. The adsorption is based on coordination between a metal ion chelated on the surface of a solid matrix and electron donor groups at the surface of the biomolecule. IMAC is a highly selective, low cost, and easily scaled-up technique being used in research and commercial operations. A separation process can be designed for a specific molecule by just selecting an appropriate metal ion, chelating agent, and operational conditions such as pH, ionic strength, and buffer type.Keywords: immobilized metal-ion affinity chromatography; purification; biomolecules. INTRODUÇÃOOs princípios fundamentais da afinidade de biomoléculas por íons metálicos são conhecidos desde o início do século passado. Em 1974, Everson e Parker demonstraram que íons metálicos presentes em metaloproteínas são os principais responsáveis pela adsorção dessas em resinas contendo quelantes imobilizados e exploraram esta afinidade na separação deste tipo de proteínas. 1 A técnica proposta por Everson e Parker popularizou-se com o trabalho publicado por Porath e colaboradores em 1975, quando os autores introduziram o termo cromatografia de afinidade por íons metálicos imobilizados (Immobilized Metal-Ion Affinity Chromatography -IMAC). 2 IMAC explora a interação entre espécies doadoras de elétrons presentes na superfície de biomoléculas em solução e íons metálicos quelatados imobilizados em um suporte sólido. Inúmeras são as aplicações analíticas, preparativas e industriais de IMAC, como a separação e purificação de diferentes biomoléculas (peptídeos, proteínas e ácidos nucléicos), a separação de células a partir de extratos biológicos e estudos de estrutura-função de proteínas.Inicialmente a técnica de IMAC foi extensamente utilizada para purificação de biomoléculas contendo naturalmente grupos doadores de elétrons em resíduos de aminoácidos expostos na superfície (tais como o anel imidazol de histidina), os quais são primariamente responsáveis pela interação biomolécula-íon metálico. Com o advento da tecnologia do DNA recombinante, foi possível a incorporação de caudas (tag) em proteínas que não contêm naturalmente espécies doadoras de elétrons, tal como a fusão de sequência de seis histidinas na porção C ou N-terminal da proteína alvo, conferindo à mesma a possibilidade de purificação por IMAC. 3,4 Apesar de na literatura existirem excelentes artigos de revisão em inglês sobre IMAC 5-14 , nesta revisão, devido à intensa atividade neste campo, pretende-se abordar aspectos fundamentais, aplicações tecnológicas (escala de laboratório e industrial) e científicas com ênfase na purificação de proteínas nativas e recombinantes (com cauda de poli(histidina)). Dentre os aspectos fundamentais do mé...

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Efficient Production of Recombinant Human (Pro)renin Utilizing a Decahistidine Tag Attached at the C-Terminus

Cited by 15 publications

References 19 publications

The Coexistence of Ser84 in Renin and His13 in Angiotensinogen Brings a pH Profile of Two Separate Peaks to the Reaction of Human Renin and Sheep Angiotensinogen

The Coexistence of Ser84 in Renin and His13 in Angiotensinogen Brings a pH Profile of Two Separate Peaks to the Reaction of Human Renin and Sheep Angiotensinogen

Qualitative and quantitative analyses of (pro)renin receptor in the medium of cultured human umbilical vein endothelial cells

Cromatografia de afinidade por íons metálicos imobilizados (IMAC) de biomoléculas: aspectos fundamentais e aplicações tecnológicas

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