Heparin Affinity Column Chromatography Research Articles

Large-Scale Preparation of Highly Stable Recombinant Human Acidic Fibroblast Growth Factor in Escherichia coli BL21(DE3) plysS Strain.

In this study, the optimum human aFGF gene encoding haFGF135 was cloned in pET3c and transferred to Escherichia coli BL21(DE3) plysS. To enhance the yield of fermentation and the expression level of the target protein, the fermentation parameters, including temperature, pH, dissolved oxygen, glucose concentration, ammonium chloride concentration, induction time, and inducer (IPTG) concentration, were optimized. The optimized fermentation parameters were used in large-scale fermentation (30 L). Ion-exchange and heparin-affinity column chromatography techniques were used for separation and purification of rhaFGF135 protein. HPLC, isoelectric focusing electrophoresis, and mass spectrometry were used to detect the purity, isoelectric point, and molecular weight and peptide map of rhaFGF135 protein, respectively. Mitogenic activity of rhaFGF135 protein was detected in NIH-3T3 cells and a full-thickness injury wound diabetic rat model. The production and expression level of rhaFGF135 in the 30-L scale fermentation reached 80.4 ± 2.7 g/L culture and 37.8% ± 1.8%, respectively. The RP-HPLC and SDS-PAGE purity of the final rhaFGF135 product almost reached 100%, and the final pure protein yield was 158.6 ± 6.8 mg/L culture. Finally, the cell and animal experiments showed that rhaFGF135 retained a potent mitogenic activity. The large-scale process of rhaFGF135 production reported herein is relatively stable and time-saving, and thus, it can be used as an efficient and economic strategy for the synthesis of rhaFGF135 at the industrial level.

Understanding the Function of a Pro-Angiogenic Polypeptide hFGF-1 with a Cancer Inhibitor Imatinib

Fibroblast growth factors (FGFs) lack signal sequences, and are exported through endoplasmic reticulum (ER)-Golgi-independent non-classical routes. FGFs work as modulators of various cellular activities like mitosis, differentiation, survival etc. Among the FGF family, which comprises of 23 different heparin proteins, human FGF-1 (hFGF-1), a potent angiogenic factors are one of the targets in cancer inhibition, as they are involved in blood vessel formation in tissues. There has been intensive research directed at the development of drugs that could effectively inhibit angiogenesis. In this context, the purpose of this study is to fully understand the molecular principles essential to determine probability of inhibition of hFGF-1 signaling transduction by imatinib. Imatinib, a 2-phenyl amino pyrimidine derivative is a tyrosine kinase inhibitor with antineoplastic activity. Imatinib binds to the intracellular pocket located within tyrosine kinases and inhibit the downstream cell proliferation events, but the exact molecular mechanism is still elusive. In this study, expression of hFGF-1 in recombinant E.coli was carried out, and the expressed protein was purified using heparin affinity column chromatography. The structural interactions governing imatinib-hFGF-1 interaction was studied by monitoring its stability, conformation and binding affinity by equilibrium unfolding using steady state fluorescence and proteolytic digestion assay. These data show that imatinib binds to hFGF-1 and enhances its thermal stability and solvent accessibility. In addition, Biacore analysis was carried out to determine the binding affinity of imatinib to hFGF-1. 1H-15N HSQC NMR was also performed in order to determine exact binding sites and stoichiometry of binding between imatinib and hFGF-1.

Asialoerythropoietin Exerts Stronger Angiogenic Activity than Erythropoietin Via its Binding Affinity to Tissue

Although erythropoietin (EPO) is known to express angiogenic and cardioprotective effects, it also induces hypertension, polycythemia, and platelet activation, which may cause serious adverse effects in patients with cardiovascular diseases. We compared the angiogenic effects of EPO and its nonerythropoietic derivative, asialo-EPO (AEPO). Lower limb ischemia was induced in ICR and C57/BL mice. Mice were injected intramuscularly with 2 μg/kg of EPO derivatives for 6 or 7 days. To assess biological differences, the tissue affinity of both EPO derivatives was analyzed in vitro using heparin affinity column chromatography. Tissue affinity was also analyzed in vivo using an intramuscular pharmacokinetic study. The survival of ischemic legs was better in the AEPO group than that in the EPO group (5/13 = 38.5 % vs 1/13 = 7.7 %, p < 0.05), and an increase in regenerated vessels was observed in the AEPO group, but not in the EPO group in ICR mice. Vessel/muscle ratios in control, EPO, and AEPO groups were 0.50 ± 0.34, 0.61 ± 0.32, and 2.83 ± 1.13, respectively (p < 0.0001). On the other hand, regenerated vessels were observed in both EPO and AEPO groups (p < 0.001) in C57/BL mice. AEPO, but not EPO, expressed heparin affinity in vitro. Intramuscularly injected EPO gradually decreased in muscle tissue, while AEPO was maintained at 2.5 ng/muscle for 1 day after several hours of a rapid clearance phase in vivo. AEPO exerts stronger angiogenic effects than those of EPO presumably via its tissue affinity. Administration of AEPO is a promising option for the treatment of patients with critical limb ischemia.

Expression of human papillomavirus 6b L1 protein in silkworm larvae and enhanced green fluorescent protein displaying on its virus-like particles

Human papillomavirus (HPV) 6b L1 capsid protein was expressed using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid expression system in silkworm larvae. Two constructs, full-length L1 (500 a.a) and C-terminal-deleted short L1 (479 a.a), and three PCR-manipulated antigenic loops at amino acids 55–56, 174–175, and 348–349 regions were incorporated with whole enhanced green fluorescent protein (EGFP). Expressed in full, short L1 proteins and variants were purified in heparin affinity column chromatography and confirmed by SDS-PAGE and western blot. The presence of self-assembled virus-like particles (VLPs) and EGFP incorporation on the surface of VLPs were confirmed by the observation of transmission electron and immunoelectron microscopies, respectively. HPV 6b L1 major capsid protein was successfully expressed in silkworm, and effective manipulation on the antigenic regions showed the path to versatile vaccine development based on HPV L1-VLPs.

Heparin Potentiates the in Vivo Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

Although bone morphogenetic proteins (BMPs) are clinically useful for bone regeneration, large amounts are required to induce new bone formation in monkeys and humans. We found recently that heparin stimulates BMP activity in vitro (Takada, T., Katagiri, T., Ifuku, M., Morimura, N., Kobayashi, M., Hasegawa, K., Ogamo, A., and Kamijo, R. (2003) J. Biol. Chem. 278, 43229-43235). In the present study, we examined whether heparin enhances bone formation induced by BMPs in vivo and attempted to determine the molecular mechanism by which heparin stimulates BMP activity using C2C12 myoblasts. Heparin enhanced BMP-2-induced gene expression and Smad1/5/8 phosphorylation at 24 h and thereafter, although not within 12 h. Heparitinase treatment did not affect the response of cells to BMP-2. In the presence of heparin, degradation of BMP-2 was blocked, and the half-life of BMP-2 in the culture medium was prolonged by nearly 20-fold. Although noggin mRNA was induced by BMP-2 within 1 h regardless of the presence of heparin, noggin failed to inhibit BMP-2 activity in the presence of heparin. Furthermore, simultaneous administration of BMP-2 and heparin in vivo dose-dependently induced larger amounts of mineralized bone tissue compared with BMP-2 alone. These findings clearly indicate that heparin enhances BMP-induced osteoblast differentiation not only in vitro but also in vivo. This study indicates that heparin enhances BMP-induced osteoblast differentiation in vitro and in vivo by protecting BMPs from degradation and inhibition by BMP antagonists.

Structural Analysis of cDNAs Coding for 4SNc-Tudor Domain Protein from Fish and Their Expression in Yellowtail Organs

We cloned complementary DNAs for 4SNc-Tudor protein (SN4TDR) from yellowtail (Seriola quinqueradiata), torafugu (Takifugu rubripes), and zebrafish (Danio rerio). This protein contains 4 staphylococcal nuclease domains at the N terminus followed by a Tudor domain. We also identified the 4SNc-Tudor proteins highly homologous to that in yellowtail from the Takifugu genomic database. According to the smart database, these fish proteins had an overlapping Tudor domain (smart00333) with a complete 5 SNc domain (smart00318). In addition, 2 possible translation start sites were observed at the 5' sequences in all 3 fish species. Northern blot analysis of different yellowtail organs showed that the full SN4TDR messenger RNA was approximately 4000 nucleotides long and that its expression was highest in liver and gallbladder, being about 2 to 5 times higher than in kidney, brain, ovary, and gills, and exceedingly low in spleen, heart, and muscle. A minor 2000-nucleotide transcript observed in kidney, spleen, and gallbladder, was attributable to an alternatively spliced variant of this gene. Total proteins extracted from yellowtail liver were fractionated by heparin affinity column chromatography and separated by sodium dodecylsulfate polyacrylamide gel electrophoresis. Analyses by SDS-PAGE and liquid chromatography with tandem mass spectroscopy identified the polypeptide encoded by SN4TDR as a single molecule of 102 kDa.

Purification and Cloning of an Endogenous Protein Inhibitor of Carp Nephrosin, an Astacin Metalloproteinase

Nephrosin is a newly discovered member of the astacin family. It is a secreted proteinase and is present in carp head kidney, kidney, and spleen, all of which are responsible for immune and hematopoietic functions in fish. A complex formed by nephrosin and its inhibitor was purified from carp kidney extract by heparin affinity column chromatography. The presence of the nephrosin-inhibitor complex in different tissues was examined by immunoblotting with polyclonal antisera against the purified nephrosin inhibitor and nephrosin. Both nephrosin and the nephrosin inhibitor were present mainly in gill, head kidney, kidney, and spleen. In addition, we have cloned the cDNA encoding the nephrosin inhibitor. There are two different cDNA clones possibly resulting from two different genes, and the long form contains unique tandem repeat sequences in the 3'-end. The deduced primary structure of nephrosin inhibitor is similar to that of fetuin-A, a mammalian protein present in blood, liver, cerebrospinal fluid, and cerebral cortex during fetal development. Treatment with both N-glycosidase F and O-glycosidase removed the carbohydrate moiety of the nephrosin inhibitor and decreased the apparent molecular mass from 40 to 30 kDa. The nephrosin inhibitor seems to be synthesized in liver and then secreted to the blood as a precursor. When it was distributed into hematopoietic tissues, it was processed from 67 to 40 kDa and acquired inhibitory activity. This processing phenomenon of fetuin has not been reported elsewhere. Importantly, the presence of an endogenous inhibitor of nephrosin is the first report of this kind for astacin enzymes. It is very likely that endogenous tissue inhibitors may also be present for the regulation of other astacin enzymes.

Characterization of two forms of acetolactate synthase from barley.

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. ALS is the target site for several classes of herbicides, including sulfonylureas, imidazolinones, and triazolopyrimidines. Two forms of ALS (designated ALS I and ALS II) were separated from barley shoots by heparin affinity column chromatography. The molecular masses of native ALS I and ALS II were determined to be 248 kDa and 238 kDa by nondenaturing gel electrophoresis and activity staining. Similar molecular masses of two forms of ALS were confirmed by a Western blot analysis. SDS-PAGE and Western blot analysis showed that the molecular masses of the ALS I and ALS II subunits were identical--65 kDa. The two ALS forms exhibited different properties with respect to the values of K(m), pI and optimum pH, and sensitivity to inhibition by herbicides sulfonylurea and imidazolinone as well as to the feedback regulation by the end-product amino acids Val, Leu, and Ile. These results, therefore, suggest that the two ALS forms are not different polymeric forms of the same enzyme, but isozymes.

Isolation and conformational analysis of fragment peptide corresponding to the heparin-binding site of hepatocyte growth factor.

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes. The mitogenic activity of HGF is mediated by its binding to a high-affinity receptor, c-Met. Heparan sulfate is an initial binding site for HGF, based on its relative abundance on the cell surface. The binding of HGF to heparin or heparin-like molecules may induce oligomerization of HGF and facilitate c-Met-dependent mitogenesis [Zioncheck et al. (1995) J. Biol.Chem. 270, 16871-16878]. Thus, heparin binding is important for the biological activity of HGF. To identify the heparin-binding site of HGF, we isolated fragment peptides corresponding to the site by limited proteolysis and chemical degradation of recombinant human HGF (rhHGF). The heparin-binding ability of the peptides was expressed as their elution positions on heparin-affinity column chromatography with NaCl gradient elution. Because all of the heparin-binding peptides obtained in this study were isolated from the N-terminal hairpin-loop region (PyrGlu32-Asn127) of HGF, the region was identified as the heparin-binding site of HGF. One of the isolated peptides, Phe42-Glu111, containing the N-terminal hairpin-loop structure, was considered a suitable model peptide for the heparin-binding site of HGF. From the observation using circular dichroism spectroscopy, it was indicated that the secondary structure of the peptide changed from a random structure to a beta-sheet-like structure upon heparin binding. In addition, oligomerization of HGF in the presence of heparin was observed by dynamic light scattering. Based on our evidence, it is considered that the conformational change in the heparin-binding site may induce the oligomerization of HGF.

Production of heparin binding epidermal growth factor-like growth factor in the early phase of regeneration after acute renal injury. Isolation and localization of bioactive molecules.

We have recently reported that heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA is induced in the rat kidney after acute ischemic injury. The present studies were designed to investigate whether bioactive HB-EGF protein is also produced in response to renal injury induced by either ischemia/reperfusion or aminoglycosides. Heparin-binding proteins were purified from kidney homogenates by heparin affinity column chromatography using elution with a 0.2-2.0 M gradient of NaCl. A single peak of proteins that eluted at 1.0-1.2 M NaCl was detected in the postischemic kidney within 6 h of injury. This eluate fraction stimulated DNA synthesis in quiescent Balb/c3T3, RIE, and NRK-52E cell lines, all of which are responsive to the epidermal growth factor family of mitogenic proteins. The EGF receptor of A431 cells was also tyrosine phosphorylated by this eluate peak. Furthermore, immunoblotting with a polyclonal antibody against rat HB-EGF indicated that the eluate peak contained immunoreactive proteins of 22 and 29 kD mol wt, consistent with the reported sizes of the secreted form and membrane anchored form of HB-EGF, respectively. Immunohistochemical studies revealed that HB-EGF was produced predominantly in distal tubules in kidneys injured either by ischemia/reperfusion or aminoglycoside administration. We also found that during metanephric development immunoreactive HB-EGF was detected in the ureteric bud as early as E14.5 and persisted in structures arising from the ureteric bud throughout embryogenesis. These results suggest that in response to acute injury, HB-EGF is produced predominantly in distal tubules and that endogenous HB-EGF may be an important growth factor involved in renal epithelial cell repair, proliferation, and regeneration in the early stages of recovery after acute renal injury, as well as in nephrogenesis.

Heparin-binding epidermal growth factor-like growth factor is an autocrine growth factor for human keratinocytes.

Since heparin-binding epidermal growth factor (HB-EGF) is a member of the EGF family and binds to EGF receptor, we tested recombinant HB-EGF for its ability to stimulate human keratinocyte proliferation. The effect of HB-EGF on human keratinocytes was dependent on the cell density. HB-EGF optimally increased the cell number by 1.8-fold at 1.0 ng/ml for a 4-day incubation period under subconfluent culture. In contrast, under confluent culture, 10 ng/ml HB-EGF optimally increased the DNA synthesis 2.1-fold. To examine the production of HB-EGF by human keratinocytes, the analysis of human keratinocyte-conditioned medium was undertaken by a combination of heparin affinity column chromatography, EGF receptor-stimulating assay, immunoblotting, and neutralization. Heparin column chromatography fractionated three activities, peaks 1, 2, and 3, which contained immunoreactive 30- and 27-, 19-, and 14.5-kDa bands, respectively. The anti-HB-EGF-blocking antibody neutralized the activities of peaks 2 and 3 by 38 and 22%, respectively, but did not neutralize the activity of peak 1 at all. The antibody reduced the cell growth by 37% for a 4-day incubation period. Northern blot analysis detected a 2.5-kilobase transcript of HB-EGF. The addition of 1 ng/ml HB-EGF optimally increased the levels of HB-EGF mRNA 5.4-fold at 1 h and TGF-alpha mRNA 3.1-fold at 3 h. Interestingly, the addition of TGF-alpha at 1 ng/ml to keratinocyte cultures enhanced the level of HB-EGF mRNA 10.2-fold at 6 h. 1 ng/ml EGF also increased HB-EGF mRNA levels 10.9-fold at 1 h. These results suggest that HB-EGF is an autocrine growth factor for human keratinocytes, and HB-EGF and TGF-alpha act not only by an autoinductive mechanism but also by mutual amplification.

Human prostate cancer model: roles of growth factors and extracellular matrices.

A human prostate cancer model was established by inoculating a prostate specific antigen (PSA)-producing LNCaP cell line with either prostate or bone fibroblasts. Alternatively, this human prostate cancer model can also be established by inoculating LNCaP cells with growth factor(s) (GFs) and extracellular matrix (ECM) immobilized on Gelfoam. The resulting LNCaP tumors were used to evaluate PSA production and excretion in athymic hosts. This model was also employed to examine the biochemical nature of mesenchymal cell-derived growth-promoting protein(s) and to assess the efficacy of potential chemotherapeutic agents. Because of the propensity of human prostate cancer to metastasize to the bone, this study defined a 1.0 M NaCl-eluted fraction, MS1, from the conditioned medium of a bone stromal cell line (MS) by heparin-affinity column chromatography. The growth-promoting activity was assayed both in vivo (e.g., tumor formation) and in vitro (e.g., soft agar colony formation). We found that the growth-promoting activity was trypsin- and heat-sensitive, and partially degraded by acid and dithiothreitol. Immunochemical studies indicated that the polyclonal antibody raised against MS1 blocked the growth-promoting effect elicited by the bone-conditioned media. This growth-promoting factor was found to be immunochemically dissimilar to KGF, HGF, and bFGF. However, addition of bFGF, HGF and NGF, but not aFGF, TGF beta, IGF1, IGF2, PDGF, EGF, TGF alpha and KGF, stimulated anchorage-independent growth of prostate cells, a condition closely parallel to tumor formation in vivo. We found that the MS1 fraction also contained fibronectin and tenascin but not laminin or collagen IV.(ABSTRACT TRUNCATED AT 250 WORDS)

Receptors for homologous plasma lipoproteins on a rat hepatoma cell line.

Hepatocytes express on their surfaces more than one class of receptors capable of mediating the internalization of lipoproteins. However, relatively little is known about the binding characteristics of hepatic receptors for various lipoproteins, about the regulation of the receptors, and about the consequences for intracellular lipid metabolism of uptake of lipoproteins via different classes of receptors. The aim of the present studies was to characterize the binding and degradation of various lipoproteins and their mutual competition for cellular processing. Since these kinds of studies may be more easily carried out in continuous established hepatoma cell lines than in nondividing primary hepatocyte cultures, we examined the lipoprotein receptor functions of a well differentiated rat hepatoma (H-35). Cells were grown to confluence in Eagle's minimal essential medium in 15% newborn calf serum. Medium then was changed to 15% lipoprotein-deficient serum for 44 hr before experiments. External binding of 125I-labeled rat plasma and intestinal lymph lipoproteins was assessed at 4 degrees C. Cellular uptake and degradation were assessed at 37 degrees C. Lipoproteins were isolated by fixed angle or zonal ultracentrifugation or by heparin affinity column chromatography and characterized as to their lipid and apoprotein compositions. Labeled low density (LDL), high density (HDL2), non-apoE-HDL, very low density lipoproteins (VLDL), and chylomicron remnants (CM-R) each manifested specific and saturable binding and degradation by the hepatoma cells. Competition experiments indicated that separate receptors were present for LDL, HDL2, and CM-R. Most of HDL2 appeared to be bound to the non-apoE-HDL receptor.(ABSTRACT TRUNCATED AT 250 WORDS)