Production of thymosin alpha1 via non-enzymatic acetylation of the recombinant precursor.
Human thymosin alpha1 is an effective immune system enhancer for the treatment of cancer and viral diseases. Therefore the development of new methods for its synthesis is an urgent problem. In the present work, we propose an efficient scalable scheme for the production of recombinant thymosin alpha1. We used an expression system based on the pET32b+ plasmid and Escherichia coli strain ER2566 to obtain a fusion protein consisting of thymosin alpha1 and thioredoxin separated by a TEV (tobacco etch virus) protease cleavage site. The fusion protein was overexpressed in soluble form and purified by ion-exchange chromatography. After proteolytic cleavage of the fusion protein with TEV protease, recombinant desacetylthymosin alpha1 was isolated by ultrafiltration. Acetic anhydride was used for selective N-terminal acetylation of the obtained peptide (yield=62%). The resultant thymosin alpha1 was purified by RP-HPLC (reversed-phase HPLC). The distinctive feature of this technology is that it is a combination of different approaches: the biotechnological production of recombinant fusion protein, its enzymatic cleavage, and chemical acetylation of desacetylthymosin alpha1. Each stage of the process was optimized to increase the yield of the target peptide, which averaged 29 mg/litre of bacterial culture. The proposed method is simple and cost-effective and is suitable for large-scale production of recombinant thymosin alpha1.
Esipov
Roman S
RS
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia. email esipov@ibch.ru
Stepanenko
Vasily N
VN
Beyrakhova
Ksenia A
KA
Muravjeva
Tatjana I
TI
Miroshnikov
Anatoly I
AI
D000107:Acetylation; D001709:Biotechnology / Q000379:methods*; D010450:Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000077596:Thymalfasin; D013947:Thymosin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D013947:Thymosin; D010450:Endopeptidases; C099488:TEV protease; D000077596:Thymalfasin