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Purification and Kinetic Properties of Human Recombinant Dihydrofolate Reductase Produced in Bombyx mori Chrysalides

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Abstract

Recent reports describe the inhibition of human dihydrofolate reductase (hDHFR) by natural tea polyphenols. This finding could explain the epidemiologic data on their prophylactic effects for certain forms of cancer, and it raises the possibility that natural and synthetic polyphenols could be used in cancer chemotherapy. In order to obtain larger quantities of hDHFR to support structural studies, we established and validated a baculovirus system for the expression of this protein in Bombyx mori chrysalides (pupae of the silkworm enclosed in a cocoon). To isolate the expressed protein, whole infected pupae were homogenized, and the expressed protein was purified by affinity chromatography. Here, we demonstrate the efficient expression of recombinant hDHFR in this model and report that this newly expressed protein has high enzymatic activity and kinetic properties similar to those previously reported for recombinant hDHFR expressed in Escherichia coli. The purified protein showed dissociation constants for the binding of natural polyphenols similar to that expressed in E. coli, which ensures its usage as a new tool for further structural studies. Although the hDHFR yield per individual was found to be lower in the chrysalides than in the larvae of B. mori, the former system was optimized as a model for the scaled-up production of recombinant proteins. Expression of proteins in chrysalides (instead of larvae) could offer important advantages from both economic and biosecurity aspects.

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Acknowledgments

This work was supported in part by grants from Fundación Séneca (Project 08595/PI/08) and Conserjería de Educación, Ciencia e Investigación (Comunidad Autónoma de Murcia; Project BIO-BMC 07/03-009). L.S.-d.-C. has a contract from the Conserjería de Educación, Ciencia e Investigación (Comunidad Autónoma de Murcia; Project BIO-BMC 07/03-009), and S.C. is contracted by the programme Torres-Quevedo from the Ministerio de Ciencia e Innovación (Spain). The authors thank Alejandro Torrecillas from the Laboratorio Integrado de Biologia Molecular (Servicio de Apoyo a la Investigacion, Universidad de Murcia) by his help in the proteomic experiments (tryptic digestion and further HPLC/MS analysis).

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Authors and Affiliations

  1. Bioprodin, S.L. Edificio CEEIM, Campus Universitario de Espinardo, Murcia, Spain

    Soledad Chazarra

  2. IMIDA, La Alberca, Murcia, Spain

    Salvador Aznar-Cervantes & José Luis Cenis

  3. Department of Biochemistry and Molecular Biology A, School of Biology, University of Murcia, Espinardo, Murcia, Spain

    Luis Sánchez-del-Campo & José Neptuno Rodríguez-López

  4. Research Unit of Clinical Analysis Service, University Hospital Virgen de la Arrixaca, Murcia, Spain

    Juan Cabezas-Herrera

  5. Collegue of Animal Sciences, Zhejiang University, Huajiachi Campus, Hangzhou, China

    Wu Xiaofeng

Authors
  1. Soledad Chazarra
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  2. Salvador Aznar-Cervantes
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  3. Luis Sánchez-del-Campo
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  4. Juan Cabezas-Herrera
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  5. Wu Xiaofeng
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  6. José Luis Cenis
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  7. José Neptuno Rodríguez-López
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Corresponding authors

Correspondence to José Luis Cenis or José Neptuno Rodríguez-López.

Additional information

Soledad Chazarra and Salvador Aznar-Cervantes have contributed equally to the work.

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Chazarra, S., Aznar-Cervantes, S., Sánchez-del-Campo, L. et al. Purification and Kinetic Properties of Human Recombinant Dihydrofolate Reductase Produced in Bombyx mori Chrysalides. Appl Biochem Biotechnol 162, 1834–1846 (2010). https://doi.org/10.1007/s12010-010-8961-9

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  • DOI: https://doi.org/10.1007/s12010-010-8961-9

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