Effective production of human growth factors in Escherichia coli by fusing with small protein 6HFh8.

BACKGROUND: Growth factors (GFs) are signaling proteins that affect cellular processes such as growth, proliferation, and differentiation. GFs are used as cosmeceuticals, exerting anti-wrinkle, anti-aging, and whitening effects, and also as pharmaceuticals to treat wounds, growth failure, and oral mucositis. However, in mammalian and bacterial cells, low productivity and expression in inclusion bodies, respectively, of GFs does not satisfy the consumer demand. Here, we aimed to develop a bacterial expression system that produces high yields of soluble GFs that can be purified in their native forms. RESULTS: We present Fh8, an 8-kDa peptide from Fasciola hepatica with an N-terminal hexa-histidine (6HFh8), as a fusion partner for enhanced human GF production in recombinant Escherichia coli. The fusion partner harboring a tobacco etch virus (TEV) protease cleavage site was fused to the N-terminus of 10 human GFs: acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), epidermal growth factor (EGF), human growth hormone (hGH), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor 165 (VEGF165), keratinocyte growth factor 1 (KGF-1), placental growth factor (PGF), stem cell factor (SCF), and tissue inhibitor of metalloproteinase 1 (TIMP-1). The fusion proteins were expressed in E. coli under the control of T7 promoter at three temperatures (25 °C, 30 °C, and 37 °C). All individual fusion proteins, except for SCF and TIMP-1, were successfully overexpressed in cytoplasmic soluble form at more than one temperature. Further, the original aFGF, IGF-1, EGF, and VEGF165 proteins were cleaved from the fusion partner by TEV protease. Five-liter fed-batch fermentation approaches for the 6HFh8-aFGF (lacking disulfide bonds) and 6HFh8-VEGF165 (a cysteine-rich protein) were devised to obtain the target protein at concentrations of 9.7 g/l and 3.4 g/l, respectively. The two GFs were successfully highly purified (> 99% purity). Furthermore, they exerted similar cell proliferative effects as those of their commercial equivalents. CONCLUSIONS: We demonstrated that 6HFh8-GF fusion proteins could be overexpressed on a g/l scale in the cytoplasm of E. coli, with the GFs subsequently highly purified and maintaining their biological activity. Hence, the small protein 6HFh8 can be used for efficient mass-production of various GFs.

Identification of novel immunogenic proteins against Streptococcus parauberis in a zebrafish model by reverse vaccinology.

Streptococcus parauberis is the major infectious agent of streptococcosis in the olive flounder (Paralichthys olivaceus), causing serious economic damage. In this study, we identified potential vaccine candidates against S. parauberis by reverse vaccinology. In total, the 2 out of 21 proteins were identified as vaccine candidates from two available S. parauberis genomes. The membrane-anchored protein SEC10/PgrA and the metal ABC transporter substrate-binding lipoprotein mtsA were potent antigenic proteins based on western blotting with mouse-derived antiserum against whole bacteria of S. parauberis serotypes I and II. In particular, metal ABC transporter substrate-binding lipoprotein (mtsA) showed similar protective immunity to that of whole-cell bacterins against S. parauberis in a zebrafish model. These results suggest that mtsA may be considered as a novel candidate in the development of vaccines against S. parauberis.

Synthesis of Fe3O4@nickel-silicate core-shell nanoparticles for His-tagged enzyme immobilizing agents.

Immobilizing enzymes on artificially fabricated carriers for their efficient use and easy removal from reactants has attracted enormous interest for decades. Specifically, binding platforms using inorganic nanoparticles have been widely explored because of the benefits of their large surface area, easy surface modification, and high stability in various pH and temperatures. Herein, we fabricated Fe3O4 encapsulated 'sea-urchin' shaped nickel-silicate nanoparticles with a facile synthetic route. The enzymes were then rapidly and easily immobilized with poly-histidine tags (His-tags) and nickel ion affinity. Porous nickel silicate covered nanoparticles achieved a high immobilization capacity (85 µg mg-1) of His-tagged tobacco etch virus (TEV) protease. To investigate immobilized TEV protease enzymatic activity, we analyzed the cleaved quantity of maltose binding protein-exendin-fused immunoglobulin fusion protein, which connected with the TEV protease-specific cleavage peptide sequence. Moreover, TEV protease immobilized nanocomplexes conveniently removed and recollected from the reactant by applying an external magnetic field, maintained their enzymatic activity after reuse. Therefore, our newly developed nanoplatform for His-tagged enzyme immobilization provides advantageous features for biotechnological industries including recombinant protein processing.

Protective efficacy of Streptococcus iniae derived enolase against Streptococcal infection in a zebrafish model.

Enolase (ENO) is one of the surface-exposed proteins of Streptococcus iniae, which previously had been identified as a plasminogen-binding protein. In this study, ENO was evaluated to induce cross-protective immunity against S. iniae and Streptococcus parauberis which are major pathogens causing streptococcosis in fish. Immunoblot analysis shows that S. iniae recombinant ENO (rENO) produced in Escherichia coli was cross-reactive with antisera against S. iniae, and S. parauberis serotype I and II. In the challenge experiment of streptococcal infection after vaccination in zebrafish, rENO elicited a similar protection with a whole cell bacterin against S. iniae and S. parauberis, which suggests its feasibility as an efficient vaccine against streptococcosis.

Retroviral gene therapy for X-linked chronic granulomatous disease: results from phase I/II trial.

X-linked chronic granulomatous disease (CGD) is an inherited immunodeficiency caused by a defect in the gp91(phox) gene. In an effort to treat X-CGD, we investigated the safety and efficacy of gene therapy using a retroviral vector, MT-gp91. Two X-CGD patients received autologous CD34(+) cells transduced with MT-gp91 after a conditioning regimen consisting of fludarabine and busulfan. The level of gene-marked cells was highest at day 21 (8.3 and 11.7% in peripheral blood cells) but decreased to 0.08 and 0.5%, respectively, 3 years after gene transfer. The level of functionally corrected cells, as determined by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase assay, reached a peak at day 17 (6.5% patient 1 (P1) and 14.3% patient 2 (P2) of total granulocytes) and declined to 0.05% (P1) and 0.21% (P2), 3 years later. Some retroviral vectors were found to have integrated within or close to the proto-oncogenes MDS1-EVI1, PRDM16, and CCND2; however, no abnormal cell expansion or related hematological malignancy was observed. Overall, the gene transfer procedure did not produce any serious adverse effects and was able to convert a significant fraction of blood cells to biologically functional cells, albeit for a short period of time.

Development of an in vitro cell culture assay system for measuring the activation of a neighbouring gene by the retroviral vector.

BACKGROUND: The use of retroviral vectors has shown an actual clinical benefit in a few inherited diseases. However, the occurrence of cases of leukemia after the X-SCID gene therapy trial raised concerns about the safety of insertional mutagenesis inherent to the biology of the retrovirus. Although the retrovirus has long been known to integrate into the host chromosome, and thus have the potential to activate the nearby gene, there has been no convenient method of studying or assaying such a cis-activation phenomenon. METHODS: In the present study, we report an in vitro assay system in which the effect of retroviral integration on the expression of the neighbouring gene can be studied. In this system, a retroviral vector and the neighbouring reporter gene were constructed in a single plasmid as if it had integrated into the chromosome. RESULTS: Using this assay, we found that the full-length long terminal repeat (LTR) could indeed activate the neighbouring gene expression from a distance and the magnitude of its activation was highly increased when this LTR was placed in the vicinity of the transcription start site of the gene, whereas the truncated LTR exerted little influence. CONCLUSIONS: This assay system might provide a useful tool for selecting the appropriate vector structure, as well as for studying the molecular mechanism underlying the cis-activation by the viral LTR.