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Published on: October 7, 2018
[Gene construction, expression and activities analysis of human leptin]
Na Wu1, Chang-Gong Zhang, Lian-Ying Xie
1School of Life Science, Xiamen University, China.
Sheng Wu Gong Cheng Xue Bao = Chinese Journal of Biotechnology
|October 14, 2006
Summary
Recombinant human leptin (rhLep) was successfully expressed and purified from E. coli. rhLep demonstrated dose-dependent effects on EC304 cells, promoting growth at low concentrations and inducing cytotoxicity and apoptosis at high concentrations.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Leptin is a key hormone regulating energy balance and metabolism.
- Understanding the biological functions of recombinant leptin is crucial for therapeutic applications.
Purpose of the Study:
- To express and purify recombinant human leptin (rhLep) in E. coli.
- To investigate the effects of rhLep on EC304 cell proliferation, viability, and apoptosis.
Main Methods:
- Gene synthesis and cloning into pET22b(+) expression vector.
- Bacterial expression in E. coli BL21 (DE3) with IPTG induction.
- Protein purification using Ni2+ affinity chromatography and identification by SDS-PAGE.
- MTT assay and AO staining to assess cell viability and apoptosis.
Main Results:
- High-yield expression of rhLep (approx. 16kD) as inclusion bodies.
- rhLep promoted EC304 cell growth at 10-30 ng/mL.
- rhLep exhibited cytotoxicity and induced massive apoptosis in EC304 cells at 50-225 ng/mL, reducing viability to 1.2% at 225 ng/mL.
Conclusions:
- The study successfully produced and characterized functional rhLep.
- rhLep exhibits dose-dependent effects on EC304 cells, highlighting its potential biological activities and cytotoxic properties.
- These findings provide a foundation for further in vitro and in vivo studies of rhLep.
