Cloning expression in E.coli and biological activity of human thymosin beta(4).
Che. Yan-Ke YK; Yang. Hui H; Lu. Fan F; Pu. Qin Q; Li. Ren-De RD; Zhao. Zhong-Liang ZL
Key Findings
- Created a recombinant plasmid that expresses thymosin‑beta‑4 in E.coli
- Achieved high expression levels, about 30% of total bacterial protein
- Purified peptide (95% pure) induced lymphocyte proliferation and differentiation in vitro
Practical Outcomes
- The work demonstrates a lab method to produce active thymosin‑beta‑4, which could interest DIY biohackers, but the protocol requires molecular‑biology skills and only shows activity in cell cultures, not human dosing or safety data.
Summary
Scientists built a DNA copy of human thymosin‑beta‑4, put it into E.coli bacteria, and got the bacteria to make a lot of the peptide. They purified it to about 95% purity and showed it can make immune cells grow and change in lab tests.
Abstract
The cDNA thymosin beta(4) was synthesized by combining of chemical and enzymatic methods. First, two complement fragments of thymosin beta(4) cDNA were synthesized by DNA synthesizer, and then denatured, annealed and extended by DNA polymerase. This fragment of thymosin beta(4) was then inserted into the EcoRV and HindIII restriction endonuclease site of an expression plasmid pLDH4 (a kind of E.coli plasmid) by blunt and cohesive ligations. Finally, the recombinant plasmid which expressed thymosin beta(4) was screened by digestion and DNA sequencing. This recombinant plasmid highly expressed the thymosin beta(4), which accounted for 30% of total bacteria proteins. By salting out and chromatography, a 95% purity of recombinant thymosin beta(4) was obtained. Biological assay indicated that the recombinant thymosin beta(4) could induce lymphocyte proliferation and differentiation.
Study Information
pubmed
2002