Email updates

Keep up to date with the latest news and content from BMC Research Notes and BioMed Central.

Open Access Research article

Expression of human protein S100A7 (psoriasin), preparation of antibody and application to human larynx squamous cell carcinoma

Manuela R Barbieri169*, Camillo DC Andrade267, Wilson A Silva267, Adriana A Marques267, Andréia M Leopoldino3, Marlise BA Montes3, Marcelo Dias-Baruffi3, Iberê C Soares4, Alda Wakamatsu4, Venâncio AF Alves4, Hélen J Laure568, Marco A Zago16 and Lewis J Greene568

Author Affiliations

1 Department of Clinical Medicine, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

2 Department of Genetics, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

3 Department of Clinical, Toxicological and Bromatological Analysis, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

4 Department of Pathology, Faculty of Medicine of São Paulo, University of São Paulo, São Paulo, SP, Brazil

5 Department of Molecular and Cellular Biology and Pathogenic Bioagents, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

6 Center for Cellular Therapy and Hemotherapy of Ribeirão Preto, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

7 Laboratory of Molecular Genetics, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

8 Protein Chemistry Center, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

9 Hemotherapy Regional Center, Center for Protein Chemistry, Tenente Catão Roxo, 2501, Monte Alegre, 14049-900, Ribeirão Preto, SP, Brazil

For all author emails, please log on.

BMC Research Notes 2011, 4:494  doi:10.1186/1756-0500-4-494

Published: 14 November 2011

Abstract

Background

Up-regulation of S100A7 (Psoriasin), a small calcium-binding protein, is associated with the development of several types of carcinomas, but its function and possibility to serve as a diagnostic or prognostic marker have not been fully defined. In order to prepare antibodies to the protein for immunohistochemical studies we produced the recombinant S100A7 protein in E. coli. mRNA extracted from human tracheal tumor tissue which was amplified by RT-PCR to provide the region coding for the S100A7 gene. The amplified fragment was cloned in the vector pCR2.1-TOPO and sub-cloned in the expression vector pAE. The protein rS100A7 (His-tag) was expressed in E. coli BL21::DE3, purified by affinity chromatography on an Ni-NTA column, recovered in the 2.0 to 3.5 mg/mL range in culture medium, and used to produce a rabbit polyclonal antibody anti-rS100A7 protein. The profile of this polyclonal antibody was evaluated in a tissue microarray.

Results

The rS100A7 (His-tag) protein was homogeneous by SDS-PAGE and mass spectrometry and was used to produce an anti-recombinant S100A7 (His-tag) rabbit serum (polyclonal antibody anti-rS100A7). The molecular weight of rS100A7 (His-tag) protein determined by linear MALDI-TOF-MS was 12,655.91 Da. The theoretical mass calculated for the nonapeptide attached to the amino terminus is 12,653.26 Da (delta 2.65 Da). Immunostaining with the polyclonal anti-rS100A7 protein generated showed reactivity with little or no background staining in head and neck squamous cell carcinoma cells, detecting S100A7 both in nucleus and cytoplasm. Lower levels of S100A7 were detected in non-neoplastic tissue.

Conclusions

The polyclonal anti-rS100A7 antibody generated here yielded a good signal-to-noise contrast and should be useful for immunohistochemical detection of S100A7 protein. Its potential use for other epithelial lesions besides human larynx squamous cell carcinoma and non-neoplastic larynx should be explored in future.

Keywords:
S100A7 (Psoriasin); Recombinant protein; Production of a polyclonal antibody; E. coli BL21::DE3; Mass spectrometry