Related Experiment Video
Updated: Aug 24, 2026
Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method
Published on: September 16, 2017
[Study on the preliminary purification and bioactivity of recombinant human TNF-alpha mutein 471]
Yao-Chen Li1, Ling-Hong Kong, Yi-Li Wang
1Institute for Cancer Research, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an 710061, China.
Aim:
To purify preliminary recombinant human TNF-alpha mutein 471 and detect its bioactivity on the basis of the TNF-alpha mutein 471 expressed in prokaryotic express system.
Methods:
The expression of recombinant human TNF-alpha mutein 471 in engineering bacteria strains E.coil was induced under the condition of optimal fermentation and expression. After cultured E.coil cells were collected and broken by using an ultrasonic disintegrator, the TNF-alpha mutein 471 existed in the form of inclusion body was extracted and purified, and then the effects of denaturation and protein concentration on protein folding were examined. The bioactivities of wild type TNF-alpha and the TNF-alpha mutein 471 were detected by MTT colorimetry.
Results:
The TNF-alpha mutein 471 was folded and polymerized successfully to from a trimer with bioactivity under the condition of proper denaturation and renaturation. The cytotoxic activity of the TNF-alpha mutein 471 to the L929 cells was 15 times as much as wild type TNF-alpha.
Conclusion:
The TNF-alpha mutein 471 expressed in prokaryotic expression system possesses significantly bioactivity after renaturation, which lays the foundation for further animal experiment and clinical experimental researches.
