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Updated: Aug 13, 2026
Identification of Protein Interacting Partners Using Tandem Affinity Purification
Published on: February 25, 2012
[The expression, renaturation, purification and preliminary identification of human IgE Cepsilon3-Cepsilon4]
Chun-xia Qiao1, Zhou Lin, Mei-ru Hu
1Department of Molecular Immunology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China. bioqcx@eyou.com
Aim:
To gain recombinant protein Cepsilon3-Cepsilon4 of IgE Fc (E34).
Methods:
We cloned the gene coding human IgE Cepsilon3-Cepsilon4 (E34) and constructed an expression vector pET28a(+)-E34. The target protein was expressed as inclusion body in E.coli BL-21. Following renaturation and purification through a CM sephorose FF column, the soluble protein was acquired, and its binding ability to murine anti-hIgE mAb was identified by Western blot and ELISA.
Results:
The cloned E34 gene was sequenced and proved by SDS-PAGE to be the same as reported sequence. SDS-PAGE analysis showed the relative molecular mass of E34 protein obtained was correct as predicted. Western blot and ELISA data revealed that it owned the epitope of binding to murine anti-hIgE mAb.
Conclusion:
The expression vector pET28a(+)-E34 has been successfully constructed and the target protein E34 recognized specifically by murine anti-hIgE mAb is obtained.
