Cloning and high expression of full length hTRF1 in E. Coli |
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Authors: | He Huang Parwaresh R Kellner U Qiao-fang Chen Jie Sun |
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Affiliation: | (1) Department of Hematology, the First Affiliated Hospital, Zhejiang University Medical College, 310003 Hangzhou, China;(2) Department of Hematology and Pathology, Kiel University, Germany |
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Abstract: | Objective: To express human telomeric repeat binding factor (TRF1) at high level in E. coli. Method: Two primers were designed
with Kpn I and EcoR I sites respectively, TRF1 cDNA fragments was amplified and cloned into plasmid pET29α, each step was
confirmed by sequencing and restriction endonuclease map analysis. And the recombinant plasmid pET29α-TRFl was then transformed
into E. coli BL21 (DE3) PlysS. Fusion protein was purified by S-protein Kit and checked by SDS-PAGE and by western blot. Result:
E. coli BL21 (DE3) PlysS expressing high level of 30 KD partial TRF1 was obtained, and TRF1 fusion protein was purified. The
optimal induction time was at 2.5 h. Excessive expression system was established and 18.6% inductive protein was obtained.
Conclusion: The expressed protein can be used for producing both polyclonal and monoclonal antibodies and for further study
of the function and structure of TRF1 and its association with malignant tumor and leukemia.
Foundation item: This work was supported by the National Natural Science Foundation of China (No. 39870339).
Biography: HUANG He (1961-), professor, the First Affiliated Hospital, Zhejiang University Medical College, majors in hematology. |
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Keywords: | TRF1 Clone Protein |
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