title: New strategy for high-level expression and purification of biologically active monomeric TGF-β1/C77S in Escherichia coli.
abstract: Mature transforming growth factor beta1 (TGF-β1) is a homodimeric protein with a single disulfide bridge between Cys77 on the respective monomers. The synthetic DNA sequence encoding the mature human TGF-β1/C77S (further termed TGF-β1m) was cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin (Trx) immediately after the DNA sequence encoding enteropeptidase recognition site. High-level expression (~1.5 g l(-1)) of Trx/TGF-β1m fusion was achieved in Escherichia coli BL21(DE3) strain mainly in insoluble form. The fusion was solubilized and refolded in glutathione redox system in the presence of zwitterionic detergent CHAPS. After refolding, Trx/TGF-β1m fusion was cleaved by enteropeptidase, and the carrier protein of TGF-β1m was separated from thioredoxin on Ni-NTA agarose. Separation of monomeric molecules from the noncovalently bounded oligomers was done using cation-exchange chromatography. The structure of purified TGF-β1m was confirmed by circular dichroism analysis. The developed technology allowed purifying biologically active tag-free monomeric TGF-β1m from bacteria with a yield of about 2.8 mg from 100 ml cell culture. The low-cost and easy purification steps allow considering that our proposed preparation of recombinant monomeric TGF-β1 could be employed for in vitro and in vivo experiments as well as for therapeutic intervention.
authors.affiliation: Department of Bioengineering, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS, 16/10 Miklukho-Maklaya, 117997, Moscow, Russia.
authors.forename: Yana V
authors.initials: YV
authors.lastname: Kim
authors.forename: Marine E
authors.initials: ME
authors.lastname: Gasparian
authors.forename: Eduard V
authors.initials: EV
authors.lastname: Bocharov
authors.forename: Rita V
authors.initials: RV
authors.lastname: Chertkova
authors.forename: Elena N
authors.initials: EN
authors.lastname: Tkach
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Dolgikh
authors.forename: Mikhail P
authors.initials: MP
authors.lastname: Kirpichnikov
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013879:Thioredoxins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D053773:Transforming Growth Factor beta1 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011993:Recombinant Fusion Proteins; D053773:Transforming Growth Factor beta1; D013879:Thioredoxins