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

• Published in: BMC research notes 2011-11, Vol.4 (1), p.494-494, Article 494
• Main Authors: Barbieri, Manuela R, Andrade, Camillo Dc, Silva, Jr, Wilson A, Marques, Adriana A, Leopoldino, Andréia M, Montes, Marlise Ba, Dias-Baruffi, Marcelo, Soares, Iberê C, Wakamatsu, Alda, Alves, Venâncio Af, Laure, Hélen J, Zago, Marco A, Greene, Lewis J
• Format: Article
• Language: English
• Subjects: Affinity chromatography; Amino acids; Antibodies; Binding proteins; Calcium-binding protein; Cell culture; Colleges & universities; Cytoplasm; E coli; E. coli BL21::DE3; Escherichia coli; Expression vectors; Gene amplification; Gene expression; Genetic aspects; Head and neck cancer; Larynx; Mass spectrometry; Mass spectroscopy; Medical research; Medicine; Messenger RNA; Molecular weight; mRNA; Nuclei; Physiological aspects; Polyclonal antibodies; Polymerase chain reaction; Production of a polyclonal antibody; Proteins; Recombinant protein; S100A7 (Psoriasin); Squamous cell carcinoma; Tumors
• ISSN: 1756-0500; 1756-0500
• DOI: 10.1186/1756-0500-4-494

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. 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. 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.