Purification of recombinant human fibroblast growth factor 13 in E. coli and its molecular mechanism of mitogenesis.

Fibroblast growth factor (FGF) 13, a member of the FGF11 subfamily, is a kind of intracrine protein similar to other family members including FGF11, FGF12, and FGF14. Unlike classical FGF, FGF13 exerts its bioactivities independent of fibroblast growth factor receptors (FGFRs). However, the effect of exogenous administration of FGF13 still remains further investigated. In the present study, we established an Escherichia coli expression system for the large-scale production of FGF13 and then obtained two isoform proteins including recombinant human FGF13A (rhFGF13A) and rhFGF13B with a purity greater than 90% by column chromatography, respectively. Otherwise, soluble analysis indicated that both rhFGF13A and rhFGF13B expressed in E. coli BL21 (DE3) pLysS were soluble. Furthermore, cellular-based experiments demonstrated that rhFGF13A, rather than rhFGF13B, could promote the proliferation of NIH3T3 cells in the presence of heparin. Mechanistically, the mitogenic effect of FGF13 was mediated by activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), but not p38. Moreover, blockage of FGFRs also significantly attenuated the mitogenic effects of rhFGF13A, implying that FGFRs are still related to FGF13. Thus, our research shows that exogenous FGF13 can act as secreted FGF to participate in cell signal transmission and heparin is still required as an ancillary cofactor for the mitogenic effects of FGF13, which may help people to discover more potential functions of FGF13 in cell life activities.

Purification of a potent mitogenic homodimeric Penicillium griseoroseum lectin and its characterisation.

Penicillium griseoroseum lectin was 80-fold purified by successive DEAE Sepharose anion exchange and Sephadex G-100 gel permeation chromatography. P. griseoroseum lectin exhibited haemagglutination activity towards protease-treated rabbit erythrocytes. It showed specificity towards various carbohydrates such as d-mannose, N-acetyl-d-glucosamine, mucins, and so forth. P. griseoroseum lectin was found as a glycoprotein with glycan content of 4.33%. Purified P. griseoroseum lectin is homodimeric having a molecular mass of 57 kDa with subunit molecular mass of 28.6 kDa. Haemagglutination activity of purified P. griseoroseum lectin was completely stable from 25°C to 35°C at a pH range of 6-7.5. Lectin activity was not influenced by divalent metal ions and denaturants. P. griseoroseum lectin manifested mitogenicity towards mice splenocytes and activity reached a peak at 75 µg/ml of lectin concentration. P. griseoroseum lectin in microgram concentrations stimulated proliferation of mice splenocytes. Thus, P. griseoroseum lectin exhibits potential mitogenicity, which can be exploited for further biomedical applications.

Solid-phase N-terminus PEGylation of recombinant human fibroblast growth factor 2 on heparin-sepharose column.

Recombinant fibroblast growth factor-2 (FGF-2) has been extensively studied and used in several clinical applications including wound healing, bone regeneration, and neuroprotection. Poly(ethylene glycol) (PEG) modification of recombinant human FGF-2 (rhFGF-2) in solution phase has been studied to increase the in vivo biostabilities and therapeutic potency. However, the solution-phase strategy is not site-controlled and the products are often not homogeneous due to the generation of multi-PEGylated proteins. In order to increase mono-PEGylated rhFGF-2 level, a novel solid-phase strategy for rhFGF-2 PEGylation is developed. RhFGF-2 proteins were loaded onto a heparin-sepharose column and the PEGylaton reaction was carried out at the N-terminus by PEG20 kDa butyraldehyde through reductive alkylation. The PEGylated rhFGF-2 was purified to near homogeneity by SP sepharose anion-exchange chromatography and the purity was more than 95% with a yield of mono-PEGylated rhFGF-2 of 58.3%, as confirmed by N-terminal sequencing and MALDI-TOF mass spectrometry. In vitro biophysical and biochemical measurements demonstrated that PEGylated rhFGF-2 has an unchanged secondary structure, receptor binding activity, cell proliferation, and MAP kinase stimulating activity, and an improved bio- and thermal stability. Animal assay showed that PEGylated rhFGF-2 has an increased half-life and reduced immunogenicity. Compared to conventional solution-phase PEGylation, the solid-phase PEGylation is advantageous in reaction time, production of mono-PEGylated protein, and improvement of biochemical and biological activity.

A B-cell mitogen from a pathogenic trypanosome is a eukaryotic proline racemase.

Lymphocyte polyclonal activation is a generalized mechanism of immune evasion among pathogens. In a mouse model of Trypanosoma cruzi infection (American trypanosomiasis), reduced levels of polyclonal lymphocyte responses correlate with resistance to infection and cardiopathy. We report here the characterization of a parasite protein with B-cell mitogenic properties in culture supernatants of infective forms, the cloning of the corresponding gene and the analysis of the biological properties of its product. We characterized the protein as a co-factor-independent proline racemase, and show that its expression as a cytoplasmic and/or membrane-associated protein is life-stage specific. Inhibition studies indicate that availability of the racemase active site is necessary for mitogenic activity. This is the first report to our knowledge of a eukaryotic amino acid racemase gene. Our findings have potential consequences for the development of new immune therapies and drug design against pathogens.

Purification and properties of an extracellular blastogen produced by group A streptococci.

An extracellular product of group A streptococci which induces lymphocyte blastogenesis has been purified to homogeneity by DEAE-cellulose and CM-cellulose chromatography. The protein, termed streptococcal blastogen A, has a mol wt of approximately or equal to 17,500 and is inactivated by protease treatment and by heating at 100 degrees C. The purified blastogen gave rise to multiple protein bands on nondenaturing polyacrylamide gel electrophoresis, only two of which possessed blastogenic activity. Treatment of the protein with dithiothreitol before electrophoresis resulted in the apparent conversion of the multiple forms to a single active species. Blastogen A differs in electrophoretic mobility from the streptococcal pyrogenic exotoxins and its lymphocyte stimulating activity is not inhibited by rabbit antisera to the exotoxins. An enzyme immunoassay has been developed to measure human antibodies against blastogen A. A selection of sera with varying levels of anti-DNase B contained antiblastogen A-IgG.

Studies on a new lymphocyte mitogen from Bordetella pertussis. I. Induction of proliferation and polyclonal antibody formation.

Pertussis B mitogen (PBM), isolated from culture supernatant fluids of Bordetella pertussis, is a potent mitogen for mouse and human lymphocytes. In mice, > 95% of the blast cells recovered from PBM cultures bear surface immunoglobulins. Therefore, PBM seems to induce proliferation of mouse B lymphocytes, but not T cells. The proliferative response observed is nonspecific because cells from all mouse strains tested, including germfree animals, are responsive. Moreover, the mitogenic activity of PBM is independent of T lymphocytes, macrophages, or serum factors. When human peripheral blood or cord blood lymphocytes are cultured in the presence of PBM, a high level of thymidine incorporation by these cells is detected. Furthermore, PBM can induce polyclonal antibody formation by both mouse and human lymphocytes. Despite similar methods of isolation, PBM is distinct from the lymphocytosis-promoting factor of B. pertussis, a previously described T cell mitogen.

A novel lectin with highly potent antiproliferative and HIV-1 reverse transcriptase inhibitory activities from cicada (Cicada flammata).

A dimeric lectin with a molecular weight of 60 kDa and high hemagglutinating activity was isolated from dried cicadas. It was adsorbed on Q-Sepharose and unadsorbed on Affi-Gel Blue gel. Its hemagglutinating activity was stable up to 55 degrees C and between pH 2 and 13. The activity was inhibited by glucuronic acid and raffinose, K(+) ions, and Mg(2+) ions. Cicada lectin potently inhibited proliferation of HepG2 hepatoma and MCF 7 breast cancer cells, with an IC(50) value of 0.76 and 0.49 microM, respectively. It potently inhibited HIV-1 reverse transcriptase activity with an IC(50) of 0.36 microM but was devoid of mitogenic activity on spleen cells. Its N-terminal sequence exhibited slight similarity to a conserved hypothetical protein from Culex quinquefasciatus and a gene product from transcript GH19834-RA of Drosophila grimshawi, but there was no resemblance to lectins from other insects, including Drosophila, Sarcophaga, Glossina, and Aedes species.

Mitogenic activity of CEL-I, an N-acetylgalactosamine (GalNAc)-specific C-type lectin, isolated from the marine invertebrate Cucumaria echinata (Holothuroidea).

An N-acetylgalactosamine (GalNAc)-specific Ca(2+)-dependent lectin (C-type lectin), isolated from the marine invertebrate Holothuroidea (Cucumaria echinata), CEL-I, showed potent mitogenic activity toward normal mouse spleen cells. The mitogenic activity of CEL-I, which reached a maximum at 100 microg/ml, was inhibited by GalNAc in a concentration-dependent manner. The mitogenic effect of CEL-I at 10 microg/ml on T cell- enriched splenocytes was at a similar level due to a well-known T cell mitogen, concanavalin A (Con A), at 10 microg/ml. Furthermore, CEL-I evoked a mitogenic response from nude mouse spleen cells, while no significant effects of Con A on this cell population were observed over a wide range of concentrations. These results suggest that CEL-I is a potent mitogenic lectin with the ability to stimulate both T and B cells.

Characterisation of a haemagglutinin from Hokkaido red bean (Phaseolus vulgaris cv. Hokkaido red bean).

BACKGROUND: A haemagglutinin was purified from Japanese Hokkaido red beans (Phaseolus vulgaris cv. Hokkaido red bean) with a procedure that included three chromatographic media. RESULTS: Haemagglutinating activity was adsorbed on DEAE cellulose, Affi-gel blue gel and Mono S. The pure haemagglutinin was a homodimer and each subunit was around 30 kDa in molecular mass. The haemagglutinating activity of this agglutinin could not be inhibited by a variety of simple sugars at 200 mmol L(-1) concentration including alpha-L-fucose, D(+)-galactose, D(+)-glucose, D(+)-glucosamine, D(-)galactosamine, galacturonic acid, (+)-lactose, D(+)-melibose, L(-)-mannose, D(+)-mannose, D-mannosamine, D(+)-raffinose, L-rhamnose, (+)-xylose and galacturonic acid. The haemagglutinating activity was fully retained at pH 4-11 and at 0-80 degrees C, but was completely lost at extreme pH values (0-2 and 13-14) and at very high temperatures (90 degrees C and 100 degrees C). The haemagglutinin exhibited a weak mitogenic activity toward mouse splenocytes, a stronger anti-proliferative activity than Con A toward HepG2 (human hepatoma) cells and inhibited >80% of HIV-1 reverse transcriptase inhibitory activity at 3.3 micromol L(-1). It was devoid of anti-fungal activity. CONCLUSION: Hokkaido red bean haemagglutinin possesses a potent anti-proliferative effect on HepG2 cells.

A Glucose binding lectin from Leucaena leucocephala seeds and its mitogenic activity against human lymphocytes.

Lectins are a specialized group of proteins with immense biological properties and applications. This study describes the purification and characterization of a lectin from Leucaena leucocephala seeds, a plant belonging to the Fabaceae family. Leucaena leucocephala lectin (LLL) was purified by a two-step purification method involving DEAE-cellulose anion exchange chromatography and Sephadex G-75 size exclusion chromatography. The isolated lectin displayed a high haemagglutination titre upon treatment with rabbit erythrocytes. SDS-PAGE and Reverse-Phase High performance liquid chromatography (RP-HPLC) analysis experimentally revealed the presence of three bands corresponding to 37, 27 and 20 kDa indicating the presence of isolectins. Periodic Acid Schiff's (PAS) staining of LLL confirmed the presence of glycoprotein. Various biochemical parameters were analysed to study its effect on the haemagglutination activity. Sugar inhibition studies experimentally revealed that Glucose was the most potent inhibitor. Fluorescence spectrometric analysis of LLL and Glucose indicated a strong interaction with an association constant of 0.159 × 103 M-1. Circular Dichroism spectroscopy indicated a higher alpha helical content (25.27%). LLL was observed to possess mitogenic activity against Peripheral blood mononuclear cells (PBMC). The present investigation reports the isolation of a novel lectin from this plant which could contribute towards the diagnostic studies of certain diseases and for its therapeutic potential.

PC12 cells as a source of neurite-derived cell surface mitogen, which stimulates Schwann cell division.

Primary cultures of rat dorsal root ganglion Schwann cells were used to assay the efficacy of PC12 cells in stimulating Schwann cell proliferation. Co-cultures of PC12 cells and Schwann cells assayed by [3H]thymidine labeling followed by autoradiography showed proliferation of Schwann cells only where contact occurred between PC12 neurites and Schwann cells. Membranes derived from PC12 cells were shown to have many characteristics similar to membranes derived from sensory neurons; both could mimic whole cells in stimulating Schwann cell division; both were inactivated by mild heat treatment and by trypsinization, and both elevated intracellular cyclic AMP concentrations in Schwann cells 16 h after addition of membranes. We conclude that PC12 cells will be a valuable source for the isolation of the neuronal cell surface component which controls proliferation of Schwann cells during development of the peripheral nervous system.

Endothelial cell mitogens derived from retina and hypothalamus: biochemical and biological similarities.

Bovine retina and hypothalamus contain anionic endothelial cell mitogens that display unusual affinities for the negatively charged glycosaminoglycan heparin. Both growth factor activities are acidic polypeptides (pl's of 5.0) as determined by isoelectric focusing and DEAE-affinity chromatography. In spite of their anionic nature, the factors bound to heparin-Sepharose columns with high affinity and could be eluted only at high salt concentrations (0.9-1.1 M NaCl). The affinity of the retina-derived growth factor (RDGF) for heparin permitted a 15,000-fold purification of the mitogen in two steps: heparin-affinity chromatography and size exclusion high-performance liquid chromatography. RDGF and the anionic hypothalamus-derived factor (aHDGF) exhibit three major biochemical similarities including isoelectric point, (pl's of 5.0), heparin affinity (elution at 0.9-1.1 M NaCl) and molecular weight (18,000). Additionally, the two factors display similar biological activities, stimulating the proliferation of capillary and human umbilical vein endothelial and 3T3 cells but not vascular smooth muscle cells. We suggest that RDGF and aHDGF are related if not identical growth factor molecules.

Mitogenic polypeptide of the mammalian seminiferous epithelium: biochemical characterization and partial purification.

A mitogenic polypeptide, previously identified in Sertoli cells of the prepuberal mouse (Feig, L. A., A. R. Bellvé, N. Horbach-Erickson, and M. Klagsbrun, 1980, Proc. Natl. Acad. Sci. USA., 77:4774-4778), now has been shown to exist in Sertoli cells of the adult mouse and in the seminiferous epithelium of several other mammalian species, including the rat, guinea pig, and calf. The levels of this seminiferous growth factor (SGF) are not appreciably reduced in adult mouse testes following hypophysectomy. SGF purified from either the adult mouse or newborn calf seminiferous epithelium has a molecular weight (Mr) of 15,700 and a pl between 4.8 and 5.8, when exposed to denaturing conditions. Furthermore, SGF from these two mammalian species probably has few exposed hydrophobic domains and has a strong propensity to aggregate into multiple, high Mr species. A purification sequence based on these biochemical properties has enabled a greater than 350-fold enrichment of SGF activity from the calf seminiferous epithelium. The protocol involves a sequence of: (a) ammonium sulfate precipitation, (b) DEAE-cellulose ion exchange chromatography, (c) gel filtration chromatography on Bio-Gel P150 in 1.0 M ammonium acetate, (d) hydrophobic chromatography on dodecyl agarose, and (e) gel filtration chromatography in 6.0 M guanidine hydrochloride. Subsequent analysis of this purified preparation by SDS PAGE, followed by silver staining, reveals approximately 7 polypeptides with Mr between 14,000 and 20,000.

Purification and characterization of a lectin from Phaseolus vulgaris cv. (Anasazi beans).

A lectin has been isolated from seeds of the Phaseolus vulgaris cv. "Anasazi beans" using a procedure that involved affinity chromatography on Affi-gel blue gel, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S, and FPLC-gel filtration on Superdex 200. The lectin was comprised of two 30-kDa subunits with substantial N-terminal sequence similarity to other Phaseolus lectins. The hemagglutinating activity of the lectin was stable within the pH range of 1-14 and the temperature range of 0-80 degrees C. The lectin potently suppressed proliferation of MCF-7 (breast cancer) cells with an IC(50) of 1.3 microM, and inhibited the activity of HIV-1 reverse transcriptase with an IC(50) of 7.6 microM. The lectin evoked a mitogenic response from murine splenocytes as evidenced by an increase in [3H-methyl]-thymidine incorporation. The lectin had no antifungal activity. It did not stimulate nitric oxide production by murine peritoneal macrophages. Chemical modification results indicated that tryptophan was crucial for the hemagglutinating activity of the lectin.

Large-scale production of biologically active human keratinocyte growth factor-2.

A rapid and efficient expression and purification system has been developed for large-scale production of biologically active recombinant human keratinocyte growth factor-2 (rhKGF-2). The gene encoding human KGF-2 was cloned into the expression vector pET3c and transformed into Escherichia coli BL21(DE3)/pLys S. Under optimal conditions in a 30-l fermentor, the average bacterial yield and the average expression level of rhKGF-2 of three batches were up to 732 g and 32%, respectively. The recombinant protein was purified by cation exchange and heparin-affinity chromatography. One hundred and sixty five milligrams of pure rhKGF-2 was achieved per liter culture. A preliminary biochemical characterization of purified rhKGF-2 was performed by Western blotting and mitogenic activity analysis, and the results demonstrated that purified rhKGF-2 could react with anti-human KGF-2 antibody and stimulate the proliferation of HaCat cells.

Purification and characterization of a mitogenic lectin from Penicillium duclauxii.

Lectins are proteins/glycoproteins of non-immune origin which interact specifically and non-covalently with carbohydrate moieties on the cell surface. In this study, a lectin was purified from Penicillium duclauxii by ion-exchange chromatography on DEAE-Sepharose and gel filtration chromatography on a Sephadex G-100 column. An overall recovery of 94.11% and 60-fold purification was achieved. The purified lectin had a molecular weight of 54.9 kDa and was found to be heterogeneous as revealed by double band of sub-units with molecular mass of 21.13 kDa and 33.26 kDa, under reducing conditions. It is a glycoprotein with carbohydrate content of 3.95%. Lectin induced haemagglutination of erythrocytes was inhibited strongly by glycoproteins such as bovine submaxillary mucin, porcine stomach mucin and fetuin. The maximum haemagglutinating activity of P. duclauxii lectin was maintained after incubation at a temperature and pH range of 20-35 °C and 6.0-8.0, respectively. The haemagglutinating activity of P. duclauxii lectin was unaffected by EDTA and various metal ions. The purified P. duclauxii lectin exhibited maximum mitogenic activity towards mouse splenocytes at a concentration of 75 µg/mL. This manuscript reports a novel lectin from P. duclauxii with potent mitogenic activity towards mouse splenocytes.

Purification and characterization of a galactose-specific lectin with mitogenic activity from pinto beans.

A galactose-specific dimeric lectin from pinto beans was purified using a procedure that involved affinity chromatography on Affi-gel blue gel, anion exchange chromatography on Q-Sepharose, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S, and FPLC-gel filtration on Superdex 200. The molecular mass of this homodimeric lectin was 62 kDa and that of each of its subunits was 31 kDa. The hemagglutinating activity of pinto bean lectin was stable within the pH range of 3-12 and the temperature range of 0-70 degrees C. By using the [3H-methyl]-thymidine incorporation assay, it was shown that the lectin had the ability to evoke a mitogenic response from murine splenocytes but it did not inhibit proliferation of L1210 leukemia cells. The pinto bean lectin inhibited HIV-1 reverse transcriptase with an IC50 of 3 microM.

Isolation, characterization, and localization of heparin-binding growth factors in the heart.

Acidic and basic fibroblast growth factors (aFGF and bFGF) are angiogenic polypeptide mitogens for cells of mesodermal and neuroectodermal origin. In this report we describe the purification from several normal human hearts (including a very fresh, nonischemic sample) of heparin-binding, acid-, heat- and trypsin-sensitive 14-18-kD peptides that crossreact with antisera against aFGF and bFGF. Further evidence includes (a) prevention of mitogenicity by protamine and by anti-bFGF, (b) displacement of 125I-bFGF from cell membranes, and (c) stimulation of capillary endothelial cell migration. Specific immunohistochemistry localized bFGF to endothelial cells and, surprisingly, to cardiac myocytes, with almost no immunoreactivity in smooth muscle cells. These peptides may function in cardiac embryogenesis, hypertrophy, atherogenesis, angiogenesis, and wound healing, and may also have endocrine, neurotropic, or vasomotor functions.

Immunogenic DNA-related factors. Nucleosomes spontaneously released from normal murine lymphoid cells stimulate proliferation and immunoglobulin synthesis of normal mouse lymphocytes.

The cell-free supernatants of normal spleen and thymus lymphocytes in short-term culture release low molecular weight (LMW) DNA protein molecules that have an immunoproliferative effect (polyclonal B cell activation) in vitro. We have determined that the protein-LMW DNA complexes responsible for these effects are nucleosomal constituents of chromatin, since the mitogenically active fractions of these cell-free supernatants contain the constituents of core histones (H3, H2A, H2B, H4) together with LMW DNA (140-180 bp), and since the immunoproliferative effects of these cell-free supernatants could be mimicked by various other nucleoprotein preparations (including calf thymus and chicken erythrocyte nucleosomes). The spontaneous cellular release of cleaved chromatin constituents in vitro can be attributed to a form of programmed cell death termed apoptosis, since the cultured spleen cells exhibited (a) morphologic evidence consistent with this process by electron microscopy, and (b) evidence of intracellular cleavage of chromatin which, like apoptosis, could be blocked with ZnSO4. This resulted in inhibition of the extracellular release of nucleosomal constituents as well as the immunoproliferative effects of the cell-free supernatants. The immunoproliferative effect of nucleosomes released from cells during apoptosis could be responsible for previously observed spontaneous in vitro anti-DNA and anti-histone antibody responses of murine spleen cells, and in vivo in normal lymphoid tissues, resulting in renewed cellular proliferation after cell death. In pathological states, this could result in abnormal polyclonal B cell proliferation and autoantibody formation.

Characteristics of prostate-derived growth factors for cells of the osteoblast phenotype.

We examined the characteristics of mitogens extracted from human benign prostatic hyperplasia and prostatic adenocarcinoma tissue. Although mitogens for fetal rat skin fibroblasts as well as for rat calvarial osteoblasts and osterosarcoma cells were found, distinct entities that acted selectively in cells of the osteoblast phenotype could be obtained by sequential reverse-phase high performance liquid chromatography. Two peptides with apparent molecular weights of 10,000 and 13,000 D were derived from hyperplastic tissue, whereas a single moiety of 10,000 D was obtained from malignant tissue. These entities increased cell numbers and alkaline phosphatase activity in osteoblastlike cells consistent with effects on both growth and differentiation. Prostatic peptides did not stimulate adenylate cyclase in osteosarcoma cells. Mitogenic activity selective for osteoblastlike cells was identified in postpubertal but not prepubertal normal prostate. The results demonstrate the existence of osteoblastic growth factors in prostatic tissue whose presence may accompany postpubertal development.