An improved strategy of TGFß3 expression in Escherichia coli: Exploiting folding modulators for a switch from misfolded to folded form.

Transforming growth factor beta 3 (TGFß3) exhibits a complex native structure featuring the presence of multiple disulfide bonds forming the active dimer. Consequently, its heterologous expression in microbial system invariably leads to inclusion body (IB) formation. In this study, we observed an interesting phenomenon of switching a significant fraction of misfolded TGFß3 to folded form by modulating the cellular protein folding machinery. We carried out co-expression experiments with chaperones and demonstrated the requirement of a coordinated action of DnaK-DnaJ-GrpE and GroESL, to achieve the native soluble conformation of TGFß3, during over-expression in E. coli. The novelty of this study lies in the fact that orchestration of a group of chaperones to work in concert for efficient folding and assembly of TGFß3-like cytokines has not been widely explored. Additionally, we have also demonstrated that presence of osmolytes (sorbitol or trehalose) in the growth media have an appreciable impact on the solubility of TGFß3. We have further shown a synergism between the effects of molecular chaperone and osmolytes on the solubility of TGFß3. We have confirmed the functionality of soluble TGFß3 by performing binding interactions with its cognate receptor TßRII. Our study delineates the fact that an effective combination of chaperones or optimum concentration of compatible osmolyte, can efficiently abrogate competing aggregation pathways and help attain the native conformation of a cysteine rich cytokine in a facile manner.

Overproduction of recombinant human transforming growth factor beta 3 in Chinese hamster ovary cells.

Transforming growth factor beta 3 (TGFß3) is an important cytokine, functioning in cell proliferation and differentiation, and has been considered to have therapeutic potential for treating various diseases and for scar reduction in adult wound healing. In the current study, a Chinese hamster ovary (CHO) cell line overexpressing recombinant human TGFß3 (rhTGFß3) was established. Through a 15-day fed-batch culture process in a 7.5-l bioreactor (5-l working volume) using chemically defined medium, the established cells could produce over 133mg/l of rhTGFß3 protein. The rhTGFß3 was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure, resulting in a recovery rate of approximately 65%, with protein purity greater than 97%. The N-terminal amino acid sequences of the purified rhTGFß3 were confirmed by N-terminal sequencing analysis. The purified rhTGFß3 was further demonstrated to be functionally active by measuring the inhibition of growth of HT-2 cells, revealing a half-maximal effective concentration of 42.11pg/ml and specific activity of 1.84×10(7)U/mg.

Identification of chemokines and growth factors in proliferative diabetic retinopathy vitreous.

Associations were investigated between levels of chemokines and growth factors in the vitreous and proliferative diabetic retinopathy (PDR). Enrolled were 58 patients (58 eyes) requiring pars plana vitrectomy (PPV), with PDR (n=32, none with traction retinal detachment) or not (non-PDR). In the latter, 16 had macular hole (MH) and 10 had epiretinal membrane (ERM). With a multiplex bead immunoassay, levels of 11 chemokines and growth factors were measured from the undiluted vitreous sample from each patient. In the non-PDR eyes, the levels of the 11 chemokines and growth factors tested were similar between patients with MH and those with ERM. However, the levels of all 11 were significantly higher in the PDR eyes relative to the non-PDR; CCL17, CCL19, and TGFß3 were markedly upregulated and have not been investigated in PDR previously. The significantly higher levels of CCL4 and CCL11 in PDR contradict the results of previous reports. Based on Spearman's nonparametric test, moderate-to-strong correlations were found between VEGF and other mediators. Our results indicate that these chemokines and growth factors could be candidates for research into targeted therapies applied either singly or in combination with anti-VEGF drugs for the treatment of PDR.

A synthetic gene increases TGFß3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule.

Human transforming growth factor-ß3 (TGFß3) is a new therapeutic protein used to reduce scarring during wound healing. The active molecule is a nonglycosylated, homodimer comprised of 13-kDa polypeptide chains linked by disulphide bonds. Expression of recombinant human TGFß3 in chloroplasts and its subsequent purification would provide a sustainable source of TGFß3 free of animal pathogens. A synthetic sequence (33% GC) containing frequent chloroplast codons raised accumulation of the 13-kDa TGFß3 polypeptide by 75-fold compared to the native coding region (56% GC) when expressed in tobacco chloroplasts. The 13-kDa TGFß3 monomer band was more intense than the RuBisCO 15-kDa small subunit on Coomassie blue-stained SDS-PAGE gels. TGFß3 accumulated in insoluble aggregates and was stable in leaves of different ages but was not detected in seeds. TGFß3 represented 12% of leaf protein and appeared as monomer, dimer and trimer bands on Western blots of SDS-PAGE gels. High yield and insolubility facilitated initial purification and refolding of the 13-kDa polypeptide into the TGFß3 homodimer recognized by a conformation-dependent monoclonal antibody. The TGFß3 homodimer and trace amounts of monomer were the only bands visible on silver-stained gels following purification by hydrophobic interaction chromatography and cation exchange chromatography. N-terminal sequencing and electronspray ionization mass spectrometry showed the removal of the initiator methionine and physical equivalence of the chloroplast-produced homodimer to standard TGFß3. Functional equivalence was demonstrated by near-identical dose-response curves showing the inhibition of mink lung epithelial cell proliferation. We conclude that chloroplasts are an attractive production platform for synthesizing recombinant human TGFß3.

An improved recombinant mammalian cell expression system for human transforming growth factor-beta2 and -beta3 preparations.

Transforming growth factor-beta2 and -beta3 (TGF-beta2 and -beta3) are important members of TGF-beta family which play important roles in the growth, maintenance, and repair processes of developing embryos, neonates, and adults. Preparation of large quantities of these two cytokines, which is necessary for structural studies and other applications, has proven to be extremely difficult. We have developed a novel Chinese hamster ovary cell-based expression system for high-level expression and high recovery of recombinant human TGF-beta2 and -beta3. In this system, we used a mammalian expression vector which contains a glutamine synthetase coding region for amplification, together with a modified TGF-beta2 or -beta3 open reading frame for expression. The leader peptide of TGF-beta2 or -beta3 was replaced by that from the V-J2-C region of a mouse immunoglobulin kappa-chain, and a poly-histidine tag was inserted immediately after the leader sequence to facilitate protein purification without changing the mature TGF-beta2 or -beta3 amino acid sequence. In addition, the extreme N-terminal cysteine residue of TGF-beta2 or -beta3 was replaced by a serine residue. The resulting expression constructs produced two stable cell clones expressing 10 mg of TGF-beta2 and 8 mg of TGF-beta3 per liter of spent medium. The purification scheme involved the use of two simple chromatographic steps with a typical yield of 5 mg of TGF-beta2 and 4 mg of TGF-beta3. This method represents a significant improvement over previously published methods and may be applicable to other TGF-beta superfamily members. We further confirmed that latent TGF-beta2 and -beta3 can be activated by proteolysis and glycolysis, which have not been reported before.