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Functional expression, characterization and application of the human S100A4 protein.

Wang D, Zhang J, Liu Z, Chen Y, Xu C, Zhang Z, Liu X, Wu L, Zhou X, Meng X, Li H, Liu H, Jiang Z, Wang T - Mol Med Rep (2014)

Bottom Line: Four hybridoma cell lines, which secreted mAbs specifically against human S100A4 protein, were obtained.One of the four mAbs, namely 2A12D10B2, recognized human S100A4 as indicated by immunohistochemical staining of human gastric carcinoma specimens and recombinant S100A4 was functionally expressed in E. coli.The mAbs of recombinant S100A4 were suitable for detecting S100A4 expression in human tissues and for investigating the subsequent clinical applications of the protein.

View Article: PubMed Central - PubMed

Affiliation: Performance Medicine Laboratory, Institute of Health and Environmental Medicine, Tianjin 300050, P.R. China.

ABSTRACT
Preparations utilizing monoclonal antibodies against S100A4 provide useful tools for functional studies to investigate the clinical applications of the human S100A4 protein. In the present study, human S100A4 protein was expressed in Escherichia coli (E. coli) BL21 (DE3), successfully purified by diethylaminoethyl cellulose anion-exchange chromatography and identified by western blot analysis. Soluble S100A4 bioactivity was confirmed by Transwell migration and invasion assays in the human HeLa cell line. Monoclonal antibodies (mAbs) were generated utilizing the standard hybridoma method and were validated by enzyme-linked immunosorbent assay and western blot analysis. The antibody was then used to examine human gastric carcinoma specimens by immunohistochemistry. Recombinant S100A4 was functionally expressed in E. coli and promoted the migration and invasion of HeLa cells. Four hybridoma cell lines, which secreted mAbs specifically against human S100A4 protein, were obtained. One of the four mAbs, namely 2A12D10B2, recognized human S100A4 as indicated by immunohistochemical staining of human gastric carcinoma specimens and recombinant S100A4 was functionally expressed in E. coli. The mAbs of recombinant S100A4 were suitable for detecting S100A4 expression in human tissues and for investigating the subsequent clinical applications of the protein.

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Recombinant expression and purification of human S100A4 protein. (A) Agarose gel analysis of human S100A4 cDNA. Lanes 1–6, human S100A4 cDNA; lane M, DNA marker. (B) Agarose gel analysis of the pET32a-S100A4 vector following restriction enzyme treatment by using Ndel and Xhol. Lanes 1–5, pET32a-S100A4 digested by Ndel and Xhol; lane 6, positive control; lane M, DNA marker. (C) DNA sequencing result. (D) Recombinant expression of pET32a-S100A4. Lane 2, positive control; lane M, protein standards; lanes 1, 3–6, 8–9, induced products of pET32a-S100A4; lane 7, negative control. (E) SDS-PAGE analysis of bacterial cultures in the sediment fraction or supernatant fraction. Lane 1, positive control; lane M, protein standards; lane 2, induced products of pET32a-S100A4 in the supernatant fraction; lane 3, induced products of pET32a-S100A4 in the sediment fraction. (F) Western blot analysis of the recombinant protein. Lane M, protein standards; lane 1, negative control; lane 2, induced products of pET32a-S100A4 in the supernatant fraction; lane 3, induced products of pET32a-S100A4 in the sediment fraction.
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f1-mmr-11-01-0175: Recombinant expression and purification of human S100A4 protein. (A) Agarose gel analysis of human S100A4 cDNA. Lanes 1–6, human S100A4 cDNA; lane M, DNA marker. (B) Agarose gel analysis of the pET32a-S100A4 vector following restriction enzyme treatment by using Ndel and Xhol. Lanes 1–5, pET32a-S100A4 digested by Ndel and Xhol; lane 6, positive control; lane M, DNA marker. (C) DNA sequencing result. (D) Recombinant expression of pET32a-S100A4. Lane 2, positive control; lane M, protein standards; lanes 1, 3–6, 8–9, induced products of pET32a-S100A4; lane 7, negative control. (E) SDS-PAGE analysis of bacterial cultures in the sediment fraction or supernatant fraction. Lane 1, positive control; lane M, protein standards; lane 2, induced products of pET32a-S100A4 in the supernatant fraction; lane 3, induced products of pET32a-S100A4 in the sediment fraction. (F) Western blot analysis of the recombinant protein. Lane M, protein standards; lane 1, negative control; lane 2, induced products of pET32a-S100A4 in the supernatant fraction; lane 3, induced products of pET32a-S100A4 in the sediment fraction.

Mentions: The gene fragment of human S100A4 was assembled by PCR. The full length of the gene fragment was 306 bp (Fig. 1A). The enzyme digestion identification and DNA sequencing confirmed that the human S100A4 gene was successfully cloned into the pET32a in the correct orientation (Figs. 1B and C). The recombinant plasmid pET32a-S100A4 was transformed to an expression host, namely, E. coli BL21 (DE3). The recombinant protein had a molecular weight of approximately 11.5 kDa, which is consistent with the estimated size (Fig. 1D; lanes 1, 3–6, 8–9). However, this band was not observed in the negative control samples (Fig. 1D; lane 7). The expressed recombinant protein was identified in the supernatant and in an insoluble form as inclusion bodies (Fig. 1E). To characterize the recombinant protein further, western blotting was performed using an goat anti-human S100A4 antibody. The recombinant protein reacted with the antibody, thus indicating the successful expression of recombinant human S100A4 protein in E. coli BL21 (DE3) cells (Fig. 1F). The presence of the recombinant protein confirmed that the recombinant human S100A4 protein was successfully purified by using ion exchange chromatography.


Functional expression, characterization and application of the human S100A4 protein.

Wang D, Zhang J, Liu Z, Chen Y, Xu C, Zhang Z, Liu X, Wu L, Zhou X, Meng X, Li H, Liu H, Jiang Z, Wang T - Mol Med Rep (2014)

Recombinant expression and purification of human S100A4 protein. (A) Agarose gel analysis of human S100A4 cDNA. Lanes 1–6, human S100A4 cDNA; lane M, DNA marker. (B) Agarose gel analysis of the pET32a-S100A4 vector following restriction enzyme treatment by using Ndel and Xhol. Lanes 1–5, pET32a-S100A4 digested by Ndel and Xhol; lane 6, positive control; lane M, DNA marker. (C) DNA sequencing result. (D) Recombinant expression of pET32a-S100A4. Lane 2, positive control; lane M, protein standards; lanes 1, 3–6, 8–9, induced products of pET32a-S100A4; lane 7, negative control. (E) SDS-PAGE analysis of bacterial cultures in the sediment fraction or supernatant fraction. Lane 1, positive control; lane M, protein standards; lane 2, induced products of pET32a-S100A4 in the supernatant fraction; lane 3, induced products of pET32a-S100A4 in the sediment fraction. (F) Western blot analysis of the recombinant protein. Lane M, protein standards; lane 1, negative control; lane 2, induced products of pET32a-S100A4 in the supernatant fraction; lane 3, induced products of pET32a-S100A4 in the sediment fraction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4237081&req=5

f1-mmr-11-01-0175: Recombinant expression and purification of human S100A4 protein. (A) Agarose gel analysis of human S100A4 cDNA. Lanes 1–6, human S100A4 cDNA; lane M, DNA marker. (B) Agarose gel analysis of the pET32a-S100A4 vector following restriction enzyme treatment by using Ndel and Xhol. Lanes 1–5, pET32a-S100A4 digested by Ndel and Xhol; lane 6, positive control; lane M, DNA marker. (C) DNA sequencing result. (D) Recombinant expression of pET32a-S100A4. Lane 2, positive control; lane M, protein standards; lanes 1, 3–6, 8–9, induced products of pET32a-S100A4; lane 7, negative control. (E) SDS-PAGE analysis of bacterial cultures in the sediment fraction or supernatant fraction. Lane 1, positive control; lane M, protein standards; lane 2, induced products of pET32a-S100A4 in the supernatant fraction; lane 3, induced products of pET32a-S100A4 in the sediment fraction. (F) Western blot analysis of the recombinant protein. Lane M, protein standards; lane 1, negative control; lane 2, induced products of pET32a-S100A4 in the supernatant fraction; lane 3, induced products of pET32a-S100A4 in the sediment fraction.
Mentions: The gene fragment of human S100A4 was assembled by PCR. The full length of the gene fragment was 306 bp (Fig. 1A). The enzyme digestion identification and DNA sequencing confirmed that the human S100A4 gene was successfully cloned into the pET32a in the correct orientation (Figs. 1B and C). The recombinant plasmid pET32a-S100A4 was transformed to an expression host, namely, E. coli BL21 (DE3). The recombinant protein had a molecular weight of approximately 11.5 kDa, which is consistent with the estimated size (Fig. 1D; lanes 1, 3–6, 8–9). However, this band was not observed in the negative control samples (Fig. 1D; lane 7). The expressed recombinant protein was identified in the supernatant and in an insoluble form as inclusion bodies (Fig. 1E). To characterize the recombinant protein further, western blotting was performed using an goat anti-human S100A4 antibody. The recombinant protein reacted with the antibody, thus indicating the successful expression of recombinant human S100A4 protein in E. coli BL21 (DE3) cells (Fig. 1F). The presence of the recombinant protein confirmed that the recombinant human S100A4 protein was successfully purified by using ion exchange chromatography.

Bottom Line: Four hybridoma cell lines, which secreted mAbs specifically against human S100A4 protein, were obtained.One of the four mAbs, namely 2A12D10B2, recognized human S100A4 as indicated by immunohistochemical staining of human gastric carcinoma specimens and recombinant S100A4 was functionally expressed in E. coli.The mAbs of recombinant S100A4 were suitable for detecting S100A4 expression in human tissues and for investigating the subsequent clinical applications of the protein.

View Article: PubMed Central - PubMed

Affiliation: Performance Medicine Laboratory, Institute of Health and Environmental Medicine, Tianjin 300050, P.R. China.

ABSTRACT
Preparations utilizing monoclonal antibodies against S100A4 provide useful tools for functional studies to investigate the clinical applications of the human S100A4 protein. In the present study, human S100A4 protein was expressed in Escherichia coli (E. coli) BL21 (DE3), successfully purified by diethylaminoethyl cellulose anion-exchange chromatography and identified by western blot analysis. Soluble S100A4 bioactivity was confirmed by Transwell migration and invasion assays in the human HeLa cell line. Monoclonal antibodies (mAbs) were generated utilizing the standard hybridoma method and were validated by enzyme-linked immunosorbent assay and western blot analysis. The antibody was then used to examine human gastric carcinoma specimens by immunohistochemistry. Recombinant S100A4 was functionally expressed in E. coli and promoted the migration and invasion of HeLa cells. Four hybridoma cell lines, which secreted mAbs specifically against human S100A4 protein, were obtained. One of the four mAbs, namely 2A12D10B2, recognized human S100A4 as indicated by immunohistochemical staining of human gastric carcinoma specimens and recombinant S100A4 was functionally expressed in E. coli. The mAbs of recombinant S100A4 were suitable for detecting S100A4 expression in human tissues and for investigating the subsequent clinical applications of the protein.

Show MeSH
Related in: MedlinePlus