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Cloning, Overexpression, Purification and Preliminary Characterization of Human Septin 8

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Abstract

Mammalian septins comprise a family of 14 genes that encode GTP-binding proteins involved in important cellular processes such as cytokinesis and exocytosis. Expression of three different constructs encoding human septin 8 were analyzed and the results show that SEPT8GC, a clone expressing the conserved domain plus C-terminal domain of human septin 8 yields the highest amount of recombinant protein. This protein was purified by affinity chromatography followed by a gel filtration chromatography. CD spectrum of SEPT8GC is characteristic of folded proteins and it presents a transition profile with a T m of 54 °C. Fluorescence emission spectra, analytic gel filtration and DLS reflect the sample oligomeric heterogeneity with the predominance of dimers in solution. Homology models indicate clearly that the preferred dimer interface is the one comprising the GTP binding site.

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Abbreviations

GTP:

Guanosine triphosphate

GDP:

Guanosine diphosphate

CD:

Circular dichroism

T m :

Melting temperature

DLS:

Dynamic light scattering

TRAFAC:

Translation factor related class

kDa:

kilodalton

SEPT2:

Human Septin 2

SEPT6:

Human Septin 6

SEPT8:

Human Septin 8

SEPT10:

Human Septin 10

SEPT11:

Human Septin 11

SEPT14:

Human Septin 14

PCR:

Polymerase Chain Reaction

IPTG:

Isopropyl-β-d-thiogalactopyranoside

FPLC:

Fast performance liquid chromatography

SEPT8I:

Construct expressing human septin 8

SEPT8GC:

Construct expressing GTPase domain plus C-terminal of human septin 8

SEPT8G:

Construct expressing GTPase domain of human septin 8

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Acknowledgments

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP grants 2005/05149-6 and 1998/14138-2) and Associação Brasileira de Tecnologia de Luz Síncrotron (ABTLuS).

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Authors and Affiliations

  1. Institute of Biology, State University of Campinas (UNICAMP), Campinas, SP, Brazil

    T. A. C. B. Souza & J. A. R. G. Barbosa

  2. Brazilian Synchrotron Light Laboratory (LNLS), C.P. 6192, 13083-970, Campinas, SP, Brazil

    T. A. C. B. Souza & J. A. R. G. Barbosa

  3. Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, SGAN 916, Módulo C, Av. W5 Norte Asa Norte, CEP: 70790-160, Brasília, DF, Brazil

    J. A. R. G. Barbosa

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  1. T. A. C. B. Souza
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Correspondence to J. A. R. G. Barbosa.

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Souza, T.A.C.B., Barbosa, J.A.R.G. Cloning, Overexpression, Purification and Preliminary Characterization of Human Septin 8. Protein J 29, 328–335 (2010). https://doi.org/10.1007/s10930-010-9256-2

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  • DOI: https://doi.org/10.1007/s10930-010-9256-2

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