Production And Characterization Of Thymosin β4in Escherichia Coli

Thymosin β4(Tβ4) is a small peptide composed of43amino acids. It has manyimportant biological functions, such as promoting cardiac repair and wound healing,and therefore has great potential in clinical applications. Previously, the preparation ofTβ4depended mainly on chemical synthesis, which is a costly process. However,genetic engineering methods have now been applied to the production of Tβ4. Therecombinant E.coli which express Tβ4in soluble form has been constructed in ourlaboratory.In this paper, the culture condition for E.coli grown in shaking flask wasoptimized. The optimized factors include inoculation amount, induction time, IPTGconcentration, temperature and cultivation time after induction. The final OD600wasabove1.5and the expression level of fusion protein above40%.The final biomass concentration of the previous fermentation process conductedin a30L bioreactor was approximately20g/L dry cell weight. In order to improve theprocess productivity, high cell density cultivation was developed in the fermentationprocess. A feedback glucose feeding strategy was used to avoid acetate accumulation.Several important factors including the temperature and pH after induction, the ratioof glucose to nitrogen in the feeding medium were optimized using orthogonalexperiment. The induction time was also optimized. The final biomass concentrationwas approximately50g/L dry cell weight. SDS-PAGE analysis demonstrated that thefusion protein was highly expressed in a soluble form, accounting for about40%ofthe total soluble protein.To obtain highly purified protein produced from high cell density cultivation, apurification process involving a three-step column procedure was implemented.Immobilized metal-ion affinity chromatography was used to purify the fusion proteinfrom the disrupted cells. Gel filtration chromatography was used to remove imidazoleprior to thrombin cleavage. Anion exchange chromatography was selected for furtherpurification. According to the results of Tricine-SDS-PAGE, the majority of theimpurities was removed. The E-rosette assay was used to determine the biologicalactivity of the Tβ4. The result demonstrated that the recombinant Tβ4was able toenhance the percentage of RFC more than10%compared with the negative control. Itindicated that the E-rosette formation capacity of lymphocytes increased after treatment with Tβ4.