Preparation Of A New Recombinant TP-5 Cyclic Analogue Cyclo-(Cys-Arg-Lys-Asp-Val-Tyr-)

Thymopoietin is a polypeptide hormone from thymus consisting of 49 amino acids. The pentapeptide thymopentin (TP-5) Arg-Lys-Asp-Val-Tyr, corresponding to the amino acids 32-36 of thymopoietin , has all the biological activities of thymopoietin as an active site of the native hormone. As a chemosynthetical linear short-peptide, TP-5 has some shortcomings such as short half-life, poor stability and weak tolerance for the proteinase in vivo, which severely limit its clinical application. In order to improve the stability and bioactivity of the peptide, a cyclic recombinant TP-5 analogue cyclo-(Cys- Arg-Lys -Asp-Val-Tyr-) [for short cTP] was designed and preprared through gene engineering's techniques.The cTP linear precursor gene encoding 6-amino acids peptide was designed, synthesized and cloned into Escherichia coli expression vector pTWIN1. Compared with the amino acid sequence of TP-5, cTP linear precursor has an extra cysteine residue at N-terminal. The recombinant vector pTW-cTP was transferred into expression E coli ER2566 cells. After induced by IPTG, a multiplex fusion protein composed of two purification tags (Chitin binding domain, CBD), intein1,intein2 and target peptide (CBD-intein1-cTP-intein2-CBD) was expressed efficiently.The growth curve of the engineered strain cTP-ER2566 was made under the laboratory condition and helped to determine the point at which the expression of the target fusion protein was induced. The optimum conditions of fermentation were explored, and the target fusion protein was induced efficiently by 0.4mmol/L IPTG for 3-4h at 30°C at the point when the A₆₀₀ of the culture reached 0.5-1.0. 1% glycerin was chosen as carbon source while glucose was founded to inhibited the expression of the target fusion protein. The fermentation with high density was then conducted in 5L fermentor following the parameters from the preliminary assays. One hundred and two grams of wet bacteria was obtained from 3L culture. The expression of target fusion protein reached 33-35% of total bacterial protein and 29% of the total soluble proteins. The fermentation of the engineered strain cTP-ER2566 ensured the further preparation of cTP.The multiplex fusion protein (CBD-intein1-cTP-intein2-CBD) was purified by...