Human thymosin alpha 1 (Talpha1) is an important peptide in the development and 
senescence of immunological competence in human, and many studies have reported 
the expression of this peptide. In this study, we designed and synthesized the 
Talpha1 gene according to the E. coli codon usage preference and constructed a 
6xTalpha1 concatemer. The latter was inserted into an E. coli expression vector 
pET-22b (+), and transformed into E. coli BL21 (DE3). After induction with IPTG, 
the concatemer protein was successfully expressed in E. coli then cleaved by 
hydroxylamine to release the Talpha1 monomer. Gly-SDS-PAGE and mass spectrometry 
confirmed that the recombinant protein was cleaved as intended. The bioactivity 
of the Talpha1 monomer was analyzed by lymphocyte proliferation and by 
mitochondrial activity in two different tumor cell lines. This study provides a 
description of the preparation of a bioactive Talpha1, which may prove useful in 
future biomedical research.