Optimization and Scaling of a Laboratory Method for Obtaining of Human Recombinant Thymosin Beta 4 up to Pilot Process

Автор: D.A. Makarov, T.I. Muraviova, V.N. Stepanenko, V.I. Shvets, and R.S. Esipov

Страница: 35-44

Optimization and Scaling of a Laboratory Method for Obtaining of Human Recombinant Thymosin Beta 4 up to Pilot Process

Russian Journal of Biotechnology, 2014, N 4, P. 35-44

UDC 573.6.086

Section:  “Biologicals Technology”

D.A. Makarov ¹,  T.I. Muraviova ¹,  V.N. Stepanenko ¹,  V.I. Shvets ²,  and  R.S. Esipov ^{1, *}

¹  The Shemyakin-and-Ovchinnikov Institute for Bioorganic Chemistry, Russ. Acad. Sci.,  117997, Moscow Russia

²  The Lomonosov Moscow University for Fine Chemical Technology,  119571, Moscow Russia

e-mail:  esipov@ibch.ru

The parameters of culturing and purification of thymosin beta 4 were optimized, and the stages of culturing and laboratory method for its isolation were scaled up to the pilot process in order to minimize the losses of the target product. Using a pilot-scale 50-l fermenter, it became possible to increase in the yield of the wet biomass from 3g to 17 g per liter of the culture broth. An additional stage of the thermal treatment of the cell lysate was introduced in the optimized process. This stage provided the 70% enrichment of the cell lysate with the target protein, which in turn permitted to enhance the efficient load on the sorbent and simplify the protein elution from a column at the stage of anion-exchanger chromatography. The ultrafiltration was substituted by the cation-chromatography; as a result, the yield of the target product at the stage of the separation of deacetyl thymosin beta 4 and thioredoxine was enhanced. The optimum conditions for the chemical deacetyl thymosin acetylation were selected; they made it possible to achieve a 93% summary yield of the reaction and to increase the practical yield of thymosin beta 4 from 3 mg to 5 mg per 1 g of biomass. The performed work resulted in the growth of the protein summary yield from 20 mg to 85 mg per 1 l of the culture.

Key words:  protein chemical acetylation,  protein isolation,  protein sedimentation,  thymosin beta 4.

23.03.2015, 1634 просмотра.