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The Expression and Purification of Recombinant Human Cardiac Troponin T Protein

Author: LvXing
Tutor: QingZhiJu
School: Central South University
Course: Clinical
Keywords: human cardiac troponin T recombinant protein prokaryoticexpression system Ni-NTA affinity ehromatography
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Type: Master's thesis
Year: 2013
Downloads: 9
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Abstract


ObjectiveTo study the expression and purification of recombinant human cardiac troponin T in escherichia coli and lay a theoretical foundation for the further preparation of high-specificity monoclonal antibody and high-stability hcTnT detection kit.Methods1. Expression of recombinant protein in escherichia coli:the fusion plasmid pET-15b-cTNNT2which contains cTnT gene fragment was expressed in E.coli BL21(DE3). And the E.coli was inoculated to Medium LB containing ampicillin. After optimizing the concentration and induction time of IPTG, we used SDS-PAGE to analyze the supernatant and pellet of the bacterial lysates. With these methods, we could evaluate whether recombinant protein was normally expressed in BL21(DE3).We could investigate the expression level of recombinant protein. And we could analyze solubility of the recombinant protein.2. Purification of recombinant protein:the N-terminus of recombinant protein contains a six histidine affinity tag which combines metal chelate to separate the recombinant protein from other one, thus the recombinant protein can be purified. In this study, the recombinant protein was bound with Ni2+after flowing through a Ni-NTA column, and other non-labeled protein outflowed along with the effluent liquid. After the elution liquid washed away most of other protein, and finally we could obtain highly purified recombinant protein.3. Identification of the purified protein by Western-blot.Results1. The fusion plasmid pET-15b-cTNNT2was transduced in E.coli BL21(DE3) successfully, which exhibited relative high expression after induction of IPTG. And the expressed recombinant protein accounted for8%of the total bacterial protein by the analysis of Bandscan5.0. High concentration of troponin T was also found in bacterial lysates through the assay of Roche Cobas E411electrochemical luminescence automatic immune analyzer.2. In this study, the recombinant protein was purified by Ni-NTA affinity ehromatography, and finally we obtained highly purified recombinant protein.3. The purified recombinant protein was analyzed by Western-blot. and the results showed that the purified protein was the recombinant hcTnT protein which we wanted.Conclusionl.The recombinant cTnT was successfully expressed in E.coli BL21(DE3)using genetic engineering technology. 2.We obtained highly purified recombinant cTnT protein using Ni-NTA affinity ehromatography.

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