LIU Ying, LI Jian-zhong, ZHANG Jun-ping. Cloning,expression,purification and subcellular localization of human ribosomal protein S3a[J]. Journal of Pharmaceutical Practice and Service, 2011, 29(2): 97-100.
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LIU Ying, LI Jian-zhong, ZHANG Jun-ping. Cloning,expression,purification and subcellular localization of human ribosomal protein S3a[J]. Journal of Pharmaceutical Practice and Service, 2011, 29(2): 97-100.
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Cloning,expression,purification and subcellular localization of human ribosomal protein S3a
- Received Date: 2010-06-09
- Rev Recd Date:
2010-09-12
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Abstract
Objective To express and purify the human ribosomal protein S3a and study the subcellular localization of human ribosomal protein S3a. Methods The RPS3a gene was obtained by RT-PCR, total RNA extracted from human HEK293 cell as the template,and cloned into the prokaryotic expressing vector PET-21a to form PET21a-RPS3a, then transferred into (Escherichia coli)BL21(DE3). PET21a-RPS3a was highly expressed in (Escherichia coli)BL21(DE3) in the presence of isopropyl-β-D-thiogalactopyranoside (IPTG) and most products existed in a soluble form. After ultrasonication, recombinant fusion proteins were purified by Ni2+-NTA affinity chromatography and 50 kD ultrafiltration membrane, the purity of RPS3a was further confirmed by Western Blot analysis. The full sequence of RPS3a gene was recombined in the downstream of the green fluorescent protein gene in the EGFP-N1 vector.The recombined pEGFP-N1-RPS3a vector was transfected into human kidney epithelial cells (293 cells), through the subcellular localization, which was observed RPS3a recombinant fluorescent protein in the cell distribution. Results The higher purity of recombinant fusion protein was obtained. Subcellular localization analysis confirmed that RPS3a recombinant fluorescent protein distributed mainly in the cytoplasm. Conclusion The recombinant fusion protein PET21a-RPS3a was successfully cloned, expressed, purified. Application of subcellular localization raises the foundation to the study on nature and function of human RPS3a protein.
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