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.

An efficient large-scale refolding technique for recovering biologically active recombinant human FGF-21 from inclusion bodies.

Fibroblast growth factor 21 (FGF-21) is an important regulator in glycolipid metabolism that is a promising drug candidate for treatment of diabetes and obesity. However, the productivity of recombinant hFGF-21 (rhFGF-21) in Escherichia coli (E. coli) is relatively low, which limits its clinical application. To meet the clinical demand and control the production cost, rhFGF-21 proteins were expressed in inclusion bodies (IBs) form in Rosetta (DE3) by high cell density fermentation in 50-L scale. Hollow fiber membrane filtration technology was used to enrich the bacteria, wash, denature and refold the IBs in the current report. The renatured proteins were purified by two-step affinity chromatography. Authenticity of the purified rhFGF-21 was confirmed by the N-and C-terminal sequence, disulfide bond composition and molecular weight analyses. Results showed that the average target protein and recovery of rhFGF-21 expressed in IBs form of three batches were more than those of the soluble form. Both the rhFGF-21 proteins from the two forms showed equal potency in improving the glucose uptake in HepG2 cells and anti-diabetic effect in db/db mice. In this study, an efficient method for preparation of FGF-21 was established. This novel process provides an important technical basis for the large-scale production of rhFGF-21.

FGF21 trafficking in intact human cells revealed by cryo-electron tomography with gold nanoparticles.

The fibroblast growth factor FGF21 was labeled with molecularly defined gold nanoparticles (AuNPs), applied to human adipocytes, and imaged by cryo-electron tomography (cryo-ET). Most AuNPs were in pairs about 80 Å apart, on the outer cell surface. Pairs of AuNPs were also abundant inside the cells in clathrin-coated vesicles and endosomes. AuNPs were present but no longer paired in multivesicular bodies. FGF21 could thus be tracked along the endocytotic pathway. The methods developed here to visualize signaling coupled to endocytosis can be applied to a wide variety of cargo and may be extended to studies of other intracellular transactions.

In-Depth Venome of the Brazilian Rattlesnake Crotalus durissus terrificus: An Integrative Approach Combining Its Venom Gland Transcriptome and Venom Proteome.

Snake venoms are complex mixtures mainly composed of proteins and small peptides. Crotoxin is one of the most studied components from Crotalus venoms, but many other components are less known due to their low abundance. The venome of Crotalus durissus terrificus, the most lethal Brazilian snake, was investigated by combining its venom gland transcriptome and proteome to create a holistic database of venom compounds unraveling novel toxins. We constructed a cDNA library from C. d. terrificus venom gland using the Illumina platform and investigated its venom proteome through high resolution liquid chromotography-tandem mass spectrometry. After integrating data from both data sets, more than 30 venom components classes were identified by the transcriptomic analysis and 15 of them were detected in the venom proteome. However, few of them (PLA2, SVMP, SVSP, and VEGF) were relatively abundant. Furthermore, only seven expressed transcripts contributed to ∼82% and ∼73% of the abundance in the transcriptome and proteome, respectively. Additionally, novel venom proteins are reported, and we highlight the importance of using different databases to perform the data integration and discuss the structure of the venom components-related transcripts identified. Concluding, this research paves the way for novel investigations and discovery of future pharmacological agents or targets in the antivenom therapy.

Pro-inflammatory cytokines and growth factors in human milk: an exploratory analysis of racial differences to inform breast cancer etiology.

BACKGROUND: Analysis of cytokines and growth factors in human milk offers a noninvasive approach for studying the microenvironment of the postpartum breast, which may better reflect tissue levels than testing blood samples. Given that Black women have a higher incidence of early-onset breast cancers than White women, we hypothesized that milk of the former contains higher levels of pro-inflammatory cytokines, adipokines, and growth factors. METHODS: Participants included 130 Black and 162 White women without a history of a breast biopsy who completed a health assessment questionnaire and donated milk for research. Concentrations of 15 analytes in milk were examined using two multiplex and 4 single-analyte electrochemiluminescent sandwich assays to measure pro-inflammatory cytokines, angiogenesis factors, and adipokines. Mixed-effects ordinal logistic regression was used to identify determinants of analyte levels and to compare results by race, with adjustment for confounders. Factor analysis was used to examine covariation among analytes. RESULTS: Thirteen of 15 analytes were detected in ≥ 25% of the human milk specimens. In multivariable models, elevated BMI was significantly associated with increased concentrations of 5 cytokines: IL-1ß, bFGF, FASL, EGF, and leptin (all p-trend < 0.05). Black women had significantly higher levels of leptin and IL-1ß, controlling for BMI. Factor analysis of analyte levels identified two factors related to inflammation and growth factor pathways. CONCLUSION: This exploratory study demonstrated the feasibility of measuring pro-inflammatory cytokines, adipokines, and angiogenesis factors in human milk, and revealed higher levels of some pro-inflammatory factors, as well as increased leptin levels, among Black as compared with White women.

Highly efficient production of functional recombinant human fibroblast growth factor 22 in E. coli and its protective effects on H2O2-lesioned L02 cells.

In the 22 member mammalian FGF family, FGF22 belongs to FGF7 subfamily, and its effects are largely confined to the brain and skin. To explore the functions of FGF22 on other tissues and develop a large-scale production of recombinant human FGF22 (rhFGF22) without a fusion tag, a plasmid encoding human FGF22 (pET3a-rhFGF22) was used to express rhFGF22 in E. coli BL21 (DE3) pLysS. A large amount of rhFGF22 inclusion body protein was obtained. A two-step denaturing method successfully solubilized rhFGF22, and it was refolded and then purified in one step via heparin affinity chromatography. A yield of 105 mg rhFGF22 with a purity of up to 95% was obtained from 100 g wet bacteria. It was found that the rhFGF22 had biological activity, since it effectively attenuated H2O2-induced human hepatic L02 cell death. Analysis by qRT-PCR and Western blot demonstrated that rhFGF22 protects L02 cells from H2O2-induced oxidative damage via suppression of mitochondrial apoptosis pathways. In conclusion, the strategy described in this paper may provide a novel means to solve the production of insoluble rhFGF22 and shine new light on its translational potential.

Middle-Molecule Uremic Toxins and Outcomes in Chronic Kidney Disease.

In patients with chronic kidney disease (CKD), uremic toxins constitute a specific nontraditional risk factor. Research in this field started in the early 1990s, and a growing body of preclinical and epidemiological evidence suggests that elevated levels of uremic toxins are associated with poor outcomes in a CKD setting. The present review focuses on a specific class of uremic toxins (the "middle molecules"), which includes well-known candidates like beta-2 microglobulin and fibroblast growth factor 23. Here, we summarize the epidemiological evidence linking the middle-molecule uremic toxin (and especially the larger ones) with hard clinical end points. Our findings highlight the urgent need for clinical trials of interventions designed to decrease levels of these middle molecules in CKD patients.

Increased production of human fibroblast growth factor 17 in Escherichia coli and proliferative activity in NIH3T3 cells.

Fibroblast growth factor 17 (FGF17) is a novel member of the FGFs family, which is essential for cell development, tissue repair, tumor growth and invasion. The aim of the current study was to obtain a high expression level of recombinant human FGF17 (rhFGF17), including soluble proteins and inclusion bodies. An optimized rhFGF17 cDNA sequence was cloned into a pET3a vector, then the pET3a­hFGF17 vector was transformed into BL21(DE3)pLysS Escherichia coli cells. Expression was induced by optimizing the conditions using isopropyl ß­D­1­thiogalactopyranoside (IPTG) and it was confirmed that a 24­h exposure to 0.8 mM IPTG at 16˚C provided the optimal condition for soluble hFGF17. Furthermore, for the inclusion bodies, the optimal condition was a 4­h exposure to 0.4 mM IPTG at 37˚C. Two forms of rhFGF17 protein were purified by heparin affinity and SP Sepharose Fast Flow chromatography. MTT assays demonstrated that the purified rhFGF17 exerted an important effect on the proliferative activity of NIH3T3 cells, although there was no significant difference when compared with standard rhFGF17. Thus, an optimal and economic expression system was created in the present study for rhFGF17 in E. coli. This expression strategy enables the preparation of sufficient and highly bioactive rhFGF17 for further investigation of underlying mechanisms.

High-yield of biologically active recombinant human fibroblast growth factor-16 in E. coli and its mechanism of proliferation in NCL-H460 cells.

Fibroblast growth factor-16 (FGF16) is a member of FGF9 subfamily, which plays key role in promoting mitosis and cell survival, and also involved in embryonic development, cell growth, tissue repair, morphogenesis, tumor growth, and invasion. However, the successful high-yield purification of recombinant human fibroblast growth factor-16 (rhFGF16) protein has not been reported. In addition, lung cancer is a major cause of cancer-related deaths, which threats people's lives and its incidence has continued to rise. Learning pathways or proteins, which involved in lung tumor progression will contribute to the development of early diagnosis and targeted therapy. FGF16 promoted proliferation and invasion behavior of SKOV-3 ovarian cancer cells, whose function may be similar in lung cancer. The hFGF16 was cloned into pET-3d and expressed in Escherichia coli BL21 (DE3) pLysS. Finally, obtained two forms of FGF16 that exhibited remarkable biological activity and the purity is over 95%, meanwhile, the yield of soluble 130 mg/100 g and insoluble 240 mg/100 g. Experiments demonstrated FGF16 could promote proliferation of NCL-H460 cells by activating Akt, Erk1/2, and p38 MAPK signaling, whereas JNK had no significant effect. In total, this optimized expression strategy enables significant quantity and activity of rhFGF16, thereby meeting its further pharmacological and clinical usages.

Expression, Purification and Characterization of Recombinant Canine FGF21 in Escherichia coli.

The canine metabolic diseases, such as obesity and diabetes, have become a worldwide problem. Fibroblast growth factor 21 (FGF21) is a potent regulator which has many biological functions relative to metabolism regulation. It suggests that FGF21 plays important roles in regulating canine metabolic diseases. To acquire the recombinant canine FGF21 (rcFGF21) in Escherichia coli, the recombinant bacteria were induced by 0.5 mM IPTG for 16 hours at 16 °C, and the rcFGF21 protein was purified by Ni-NTA. 8 mg rcFGF21 was acquired from one liter bacteria. The rcFGF21 protein has specific immunoblot reactivity against anti-FGF21 and anti-His antibody. The in vivo experimental result showed that rcFGF21 can significantly reduce plasma glucose of STZ-induced diabetic mice.

Genome-wide identification, phylogeny, and expression of fibroblast growth genes in common carp.

Fibroblast growth factors (FGFs) are a large family of polypeptide growth factors, which are found in organisms ranging from nematodes to humans. In vertebrates, a number of FGFs have been shown to play important roles in developing embryos and adult organisms. Among the vertebrate species, FGFs are highly conserved in both gene structure and amino-acid sequence. However, studies on teleost FGFs are mainly limited to model species, hence we investigated FGFs in the common carp genome. We identified 35 FGFs in the common carp genome. Phylogenetic analysis revealed that most of the FGFs are highly conserved, though recent gene duplication and gene losses do exist. By examining the copy number of FGFs in several vertebrate genomes, we found that eight FGFs in common carp have undergone gene duplications, including FGF6a, FGF6b, FGF7, FGF8b, FGF10a, FGF11b, FGF13a, and FGF18b. The expression patterns of all FGFs were examined in various tissues, including the blood, brain, gill, heart, intestine, muscle, skin, spleen and kidney, showing that most of the FGFs were ubiquitously expressed, indicating their critical role in common carp. To some extent, examination of gene families with detailed phylogenetic or orthology analysis verified the authenticity and accuracy of assembly and annotation of the recently published common carp whole genome sequences. Gene families are also considered as a unique source for evolutionary studies. Moreover, the whole set of common carp FGF gene family provides an important genomic resource for future biochemical, physiological, and phylogenetic studies on FGFs in teleosts.

[Oncogenic osteomalacia. Report of two cases]. / Osteomalacia oncogénica. Presentación de dos casos.

Oncogenic osteomalacia is a rare disease. It is caused by a tumor that produces fibroblast growth factor 23, a hormone that decreases the tubular phosphate reabsorption and impairs renal hydroxylation of vitamin D. This leads to hyperphosphaturia with hypophosphatemia and low calcitriol levels. About 337 cases have been reported and we studied two cases; 44 and 70 year-old men who sought medical attention complaining of suffering diffuse bone pain over a period of approximately one year. In both cases, a laboratory test showed biochemical alterations compatible with a hypophosphatemic osteomalacia. In the first case, a soft tissue tumor of the right foot was removed, one year after the diagnosis. The patient was allowed to diminish the phosphate intake, but symptoms reappeared at this time. Eight years later, a local recurrence of the tumor was noted. A complete excision was now performed. The patient was able to finally interrupt the phosphate intake. In the second case, an F-18 fluorodeoxyglucose positron emission tomography, with computed tomography revealed a 2.26 cm diameter hypermetabolic nodule in the soft tissue of the right forefoot. After its removal, the patient discontinued the phosphate intake. Both patients are asymptomatic and show a regular phosphocalcic laboratory evaluation. The histopathological diagnosis was, in both cases, a phosphaturic mesenchymal tumor, a mixed connective tissue variant. This is the prototypical variant of these tumors.

Osteomalacia oncogénica: Presentación de dos casos

La osteomalacia oncogénica es una enfermedad rara. Existen descriptos alrededor de 337 casos. Es ocasionada por un tumor productor del factor de crecimiento fibroblástico 23 (FGF-23), hormona que disminuye la reabsorción tubular de fosfatos y altera la hidroxilación renal de la vitamina D, con hipofosfatemia, hiperfosfaturia y niveles bajos de calcitriol. Se presentan dos pacientes de 44 y 70 años, que consultaron por dolores óseos generalizados de aproximadamente un año de evolución en los que se hallaron alteraciones bioquímicas compatibles con osteomalacia hipofosfatémica. En el primer caso se realizó la resección de una tumoración en tejido celular subcutáneo del pie derecho, un año después del diagnóstico clínico. Luego de la exéresis, se disminuyó el aporte de fosfatos que recibía el paciente, pero reaparecieron los dolores al intentar suspenderlos. Ocho años más tarde, hubo recidiva local de la tumoración por lo que se efectuó resección completa. Después de la misma, se logró suspender el aporte de fosfatos. En el segundo caso, el paciente se estudió con tomografía por emisión de positrones con 18F-fluorodesoxiglucosa, hallando formación nodular hipermetabólica en partes blandas de antepie derecho, de 2.26 cm de diámetro. Luego de su escisión se pudo suspender el aporte de fosfatos. Ambos pacientes se encuentran asintomáticos con indicadores de metabolismo fosfocálcico normales. El diagnóstico anatomopatológico en ambos fue un tumor mesenquimático fosfatúrico, variante mixta del tejido conectivo, la entidad más frecuentemente asociada a la osteomalacia oncogénica.

Oncogenic osteomalacia is a rare disease. It is caused by a tumor that produces fibroblast growth factor 23, a hormone that decreases the tubular phosphate reabsorption and impairs renal hydroxylation of vitamin D. This leads to hyperphosphaturia with hypophosphatemia and low calcitriol levels. About 337 cases have been reported and we studied two cases; 44 and 70 year-old men who sought medical attention complaining of suffering diffuse bone pain over a period of approximately one year. In both cases, a laboratory test showed biochemical alterations compatible with a hypophosphatemic osteomalacia. In the first case, a soft tissue tumor of the right foot was removed, one year after the diagnosis. The patient was allowed to diminish the phosphate intake, but symptoms reappeared at this time. Eight years later, a local recurrence of the tumor was noted. A complete excision was now performed. The patient was able to finally interrupt the phosphate intake. In the second case, an F-18 fluorodeoxyglucose positron emission tomography, with computed tomography revealed a 2.26 cm diameter hypermetabolic nodule in the soft tissue of the right forefoot. After its removal, the patient discontinued the phosphate intake. Both patients are asymptomatic and show a regular phosphocalcic laboratory evaluation. The histopathological diagnosis was, in both cases, a phosphaturic mesenchymal tumor, a mixed connective tissue variant. This is the prototypical variant of these tumors.

High-efficiency production of bioactive recombinant human fibroblast growth factor 18 in Escherichia coli and its effects on hair follicle growth.

Using fusion tags, expression of recombinant human fibroblast growth factor 18 (rhFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, including chondrogenesis and osteogenesis, hair growth, and neuroprotection. However, high-level rhFGF18 expression is difficult and the products are often not homogeneous. Furthermore, fusion-tagged protein has higher immunogenicity and lower bioactivity, and the removal of the fused tag is expensive. To overcome the limitations of fusion-tagged expression of protein and to prepare soluble highly bioactive rhFGF18, we have developed a rapid and efficient expression strategy. Optimized hFGF18 gene was amplified by polymerase chain reaction and cloned into pET22b and pET3c vectors, then transformed into E. coli strains Origima (DE3) and BL21 (DE3)PlysS. The best combination of plasmid and host strain was selected, and only Origima (DE3)/pET3c-rhFGF18 was screened for high-level expressed rhFGF18. Under optimal conditions in a 30-L fermentor, the average bacterial yield and expression level of rhFGF18 of three batches were more than 652 g and 30 % respectively, after treatment with 1 mM isopropyl-thio-ß-galactopyranoside for 10 h at 25 °C. The target protein was purified by CM Sepharose FF and heparin affinity chromatography. The purity of rhFGF18 was shown by HPLC to be higher than 95 %, and the yield was 155 mg/L. In vitro MTT assays demonstrated that the purified rhFGF18 could stimulate significant proliferation of NIH3T3 cells, and animal experiments showed that rhFGF18 could effectively regulate hair growth. In conclusion, this may be a better method of producing rhFGF18 to meet the increasing demand in its pharmacological application.

SUMO fusion system facilitates soluble expression and high production of bioactive human fibroblast growth factor 23 (FGF23).

As a key humoral regulator of phosphate homeostasis and its involvement in the pathogenesis of human disease, human fibroblast growth factor 23 (hFGF23) has become a particularly attractive therapeutic target. To prepare soluble and bioactive recombinant human FGF23 to meet the increasing demand in its pharmacological application, small ubiquitin-related modifier (SUMO)-FGF23 fusion gene and FGF23 non-fusion gene were amplified by standard PCR methods and cloned into vector pET-22b and pET-3c, then transformed into Escherichia coli Rosetta (DE3) and BL21 (DE3). The best combination of plasmid and host strain was screened, and only Rosetta (DE3)/pET-SUMO-FGF23 was screened for rhFGF23 protein expressed. The average bacterial yield and the soluble expression level of recombinant hFGF23 of three batches attained 687 ± 18 g and 30 ± 1.5%, respectively, after treatment with 0.4 mM isopropyl-thio-ß-galactopyranoside for 19 h at 16 °C in a 30-L fermentor, after which it was purified by DEAE Sepharose FF and nickel nitrilotriacetic acid affinity chromatography. Once cleaved by the SUMO protease, the recombinant human FGF23 was released from the fusion protein. The purity of rFGF23 was shown by high performance liquid chromatography to be greater than 90% and the yield was 60 ± 1.5 mg/L. In vitro data showed that the purified rFGF23 can induce the phosphorylation of mitogen-activated protein kinases in the glioma U251 cell. The results of in vivo animal experiments also showed that rFGF23 could decrease the concentration in the plasma of normal rats fed with a fixed formula diet.

Large-scale expression, purification, and glucose uptake activity of recombinant human FGF21 in Escherichia coli.

As a novel important regulator of glucose and lipid metabolism homeostasis, human fibroblast growth factor 21 (hFGF21) has become a potential drug candidate for the treatment of metabolic diseases including obesity, and type 2 diabetes, as well as non-alcoholic fatty liver disease. To improve the production of recombinant hFGF21 to meet the increasing demand in clinical applications, an artificial gene encoding its mature peptide sequence was constructed, cloned into vector pET-3c and then expressed in Escherichia coli Origami B (DE3). Under optimal conditions in a 50-L fermentor, the average bacterial yield and the soluble expression level of recombinant hFGF21 of six batches attained 1750 ± 185 g and 32 ± 1.5%, respectively. The target protein was purified by the combination of nickel-nitrilotriacetic acid affinity chromatography and Sephadex S-100 resin. 5% (w/v) trehalose solution was able to prevent rhFGF21 from degradation effectively. The purity of rhFGF21 was higher than 97%, and the yield was 213 ± 17 mg/L. The preliminary biochemical characterization of rhFGF21 was confirmed using Western blot and peptide map finger analysis. Based on the glucose oxidase-peroxidase assay, the EC50 of glucose uptake activity of the purified rhFGF21 was 22.1 nM.

Expression of soluble proteins in Escherichia coli by linkage with the acidic propiece of eosinophil major basic protein.

An expression method has been developed to produce soluble cationic polypeptides in Escherichia coli while avoiding inclusion body deposition. For this technique the recombinant product is linked through a thrombin or factor Xa susceptible bond to the amino-terminal domain of the precursor of eosinophil major basic protein (MBP). This N-terminal domain is strongly acidic and is apparently able to shield eosinophils from the potentially injurious activities of MBP. It was reasoned that constructs of this acidic domain with small heterologous cationic proteins expressed in E. coli could result in soluble expression while preventing trafficking and packaging into insoluble inclusion bodies. This has been demonstrated using four examples: complement C5a, CCL18, fibroblast growth factor-ß, and leukemia inhibitory factor, whose isoelectric points range from 8.93 to 9.59. Further general applicability of this technique has been shown by using two different expression systems, one which encodes an amino-terminal oligo-histidine leash, and another that codes for an amino-terminal glutathione-S-transferase. Thus the utility of coupling MAP to cationic polypeptides for the purpose of soluble heterologous protein expression in E. coli has been demonstrated.

High-level expression and purification of soluble recombinant FGF21 protein by SUMO fusion in Escherichia coli.

BACKGROUND: Fibroblast growth factor 21 (FGF21) is a promising drug candidate to combat metabolic diseases. However, high-level expression and purification of recombinant FGF21 (rFGF21) in Escherichia coli (E. coli) is difficult because rFGF21 forms inclusion bodies in the bacteria making it difficult to purify and obtain high concentrations of bioactive rFGF21. To overcome this problem, we fused the FGF21 with SUMO (Small ubiquitin-related modifier) by polymerase chain reaction (PCR), and expressed the fused gene in E. coli BL21(DE3). RESULTS: By inducing with IPTG, SUMO-FGF21 was expressed at a high level. Its concentration reached 30% of total protein, and exceeded 95% of all soluble proteins. The fused protein was purified by DEAE sepharose FF and Ni-NTA affinity chromatography. Once cleaved by the SUMO protease, the purity of rFGF21 by high performance liquid chromatography (HPLC) was shown to be higher than 96% with low endotoxin level (<1.0 EU/ml). The results of in vivo animal experiments showed that rFGF21 produced by using this method, could decrease the concentration of plasma glucose in diabetic rats by streptozotocin (STZ) injection. CONCLUSIONS: This study demonstrated that SUMO, when fused with FGF21, was able to promote its soluble expression of the latter in E. coli, making it more convenient to purify rFGF21 than previously. This may be a better method to produce rFGF21 for pharmaceutical research and development.

Glycosaminoglycan affinity of the complete fibroblast growth factor family.

BACKGROUND: Many fibroblast growth factor family proteins (FGFs) bind to the heparan sulfate/heparin (HP) subtypes of sulfated glycosaminoglycans (GAGs), and a few have recently been reported to also interact with chondroitin sulfate (CS), another sulfated GAG subtype. METHODS: To gain additional insight into this interaction, we prepared all currently known FGFs (i.e., FGF1-FGF23) and assessed their affinity for HP, CS-B, CS-D and CS-E. In addition, midkine, hepatocyte growth factor and pleiotrophin were studied as other known HP-binding proteins. RESULTS: We found that members of the FGF19 subfamily (i.e., FGF15, 19, 21 and 23) had little or no affinity for HP; all of the other secretable growth factors tested had strong affinities for HP, as was indicated by the finding that their elution from HP-Sepharose columns required 1.0-1.5 M NaCl. We also found that FGF3, 6, 8 and 22 had strong affinities for CS-E, while FGF5 had a moderate affinity for CS-D. The interactions between FGFs and GAGs thus appear to be more diverse than previously understood. GENERAL SIGNIFICANCE: This is noteworthy, as the differential interactions of these growth factors with GAGs may be key determinants of their specific biological activities.

High-level expression and purification of Tat-haFGF19-154.

Human acidic fibroblast growth factor (haFGF) stimulates repair and regeneration of central and peripheral nerves after various injuries. However, it is unable to cross the blood-brain barrier (BBB). To produce a therapeutic haFGF with cell-permeable activity, we fused the haFGF(19-154) gene with Tat-PTD. After its construction by a single-step insertion of a polymerase chain reaction (PCR)-amplified coding sequence, the vector pTat-haFGF(19-154)-His was expressed in Escherichia coli BL21 (DE3) cells. The optimal expression level of the soluble fusion protein was up to 36.7% of the total cellular protein. The recombinant Tat-haFGF(19-154)-His was purified by a combination of Ni-NTA affinity, Sephadex G-25, and heparin affinity chromatography to 95% as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The final yield was 171 mg/l culture. Purified Tat-haFGF(19-154)-His had distinct mitogenic activity in Balb/c 3T3 cells, as measured by methylthiazoletetrazolium (MTT) assay and its ED(50) was 3.931 x 10(-4) micromol/l. Tat-haFGF(19-154)-His protein intravenously injected at the dose of 10 mg/kg could be detected in the pallium and hippocampi.