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The Protein Journal

, Volume 29, Issue 5, pp 328–335 | Cite as

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

  • T. A. C. B. Souza
  • J. A. R. G. Barbosa
Article

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.

Keywords

SEPT8 Septin 8 Purification Expression Cell cycle Homo sapiens 

Abbreviation list

GTP

Guanosine triphosphate

GDP

Guanosine diphosphate

CD

Circular dichroism

Tm

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

Notes

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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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • T. A. C. B. Souza
    • 1
    • 2
  • J. A. R. G. Barbosa
    • 1
    • 2
    • 3
  1. 1.Institute of BiologyState University of Campinas (UNICAMP)CampinasBrazil
  2. 2.Brazilian Synchrotron Light Laboratory (LNLS)CampinasBrazil
  3. 3.Pós-Graduação em Ciências Genômicas e BiotecnologiaUniversidade Católica de BrasíliaBrasíliaBrazil

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