Chemical and biological characterization of low-molecular-weight human skeletal growth factor

Skeletal growth factor (SGF) activity was extracted from human bone matrix by demineralization and purified under dissociative conditions using hydroxyapatite, HPLC gel-filtration and HPLC reverse-phase chromatography. Human SGF thus purified was characterized chemically and biologically. Purified human SGF stimulated chick embryo bone cell proliferation at picomolar concentrations (half maximum at 2-3 ng/ml) and had little or no activity on other cell types tested (mouse 3T3 and normal rat kidney fibroblasts, embryonic chick intestinal and human placental cells). Human SGF did not displace 125I-labeled epidermal growth factor binding to normal rat kidney cells and did not stimulate normal rat kidney cell colony formation in soft agar. Human SGF activity was sensitive to trypsin, chymotrypsin, papain, dithiothreitol and performic acid but was resistant to heat (upto 70 degrees C), pH (3-10), cyanogen bromide, alkaline phosphatase and neuraminidase and did not bind jack bean concanavalin A or kidney bean lectin. From our chemical and biological studies it appears that human SGF is different from other known polypeptide growth factors: epidermal growth factor, fibroblast growth factor, insulin, insulin-like growth factor-I, platelet-derived growth factor and transforming growth factor.     

Primary structure of human skeletal growth factor: homology with human insulin-like growth factor-II

Human skeletal growth factor (human SGF) extracted from human bone has been purified to homogeneity by hydroxyapatite chromatography and gel filtration under dissociative conditions followed by FPLC heparin-Sepharose affinity chromatography and reverse phase HPLC. Human SGF was homogeneous except that in each preparation about 30% of SGF molecules lacked the N-terminal alanine. 75% of the human SGF sequence has been determined. The amino acid sequences of the N-terminal 20 amino acids and of several tryptic fragments were identical to the corresponding sequences of human insulin-like growth factor-II (IGF-II) purified from serum. However, since the C-peptide (variable region) of human SGF has not yet been sequenced, we cannot conclude that SGF is identical to IGF-II. Comparison of the amino acid sequence of human SGF with that of IGF-II variants that have been described in the literature revealed that human SGF is not one of the known IGF-II variants. IGF-I was also found in human bone extract but was several-fold less abundant than SGF/IGF-II. The relative abundance of SGF/IGF-II and IGF-I in bone corresponded to the relative rates of production of these two mitogens by human bone cells in vitro. Regarding the physiological significance of IGF-II in bone, previous studies on the biological actions of SGF in vitro suggest that this growth factor can have both paracrine and autocrine functions on cells of the osteoblast line. In addition, we have proposed the concept that SGF is a mediator of the coupling of bone formation to bone resorption, an important bone volume regulatory mechanism. In as much as SGF is very similar (if not identical) to IGF-II, it seems likely that these proposed regulatory functions of SGF in bone are attributable to IGF-II.     

Purification and characterisation of a growth factor from porcine bone

A growth factor was extracted from porcine bone matrix by demineralisation and purified by heat and acid treatment, hydroxyapatite chromatography and gel filtration under dissociative conditions and reverse-phase HPLC. Using the mitogenic response of osteoblast-progenitor cells from embryonic chicken, a mitogenic activity was purified 3000-fold. The mitogenic protein thus purified shows an apparent molecular mass of 13.5 kDa in both the nonreduced and reduced form on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The mitogenic activity is sensitive to proteinase K, dithiothreitol, and resistant to DNAse, RNase, heat (70 degrees C) and pH (3-10). The factor stimulates the proliferation of osteoblast-progenitor cells from embryonic chick at a concentration of 1 ng/ml. It is active on cells from skin, periosteum and sternum and has no or little activity on cells of the calvaria, intestine or kidney of embryonic chick or on mouse AKR-2B/Balb c/3T3 cell line.     

Purification and Characterization of a Glycine-rich Protein from the Aleurone Layer of Soybean Seeds

Glycine-rich protein (GRP) was extracted with hot water from cell walls of the aleurone layer of soybean seeds and solubilized by pectinase treatment. GRP was purified by Sephadex G-100 gel chromatography, anion exchange HPLC, and reverse-phase HPLC. Two GRP fractions that had almost the same amino acid composition were found by gel chromatography. The high-molecular-mass G RP seems to be an associated form of the low-molecular-mass GRP (30-kDa). Thirty-kDa GRP was separated into a major GRP-I and a minor GRP-II by anion exchange HPLC. The major amino acids of GRP-I purified by reverse-phase HPLC were glycine (68%) and serine (12%). GRP-I contained a small proportion of sugar, approximately 9% (w/w), and mannose, arabinose, glucose, xylose, and galactose were included in the sugar moiety. The N-terminal amino acid sequence of GRP-I was a novel polyglycine structure including at least 20 glycine-repeated sequence. The GRP-I might be a novel type of extracellular matrix protein specific to the aleurone layer.     

A rapid method for the preparation of 125I-labelled human growth hormone for receptor studies, using reverse-phase high performance liquid chromatography

Human growth hormone was labelled with 125 Iodine by the stoichiometric modification of the chloramine-T method to a specific activity of 50-80 microCi/microgram, and the iodinated mixture was purified by reverse-phase high performance liquid chromatography using a C18 column (SynChropak RP-P) and a linear gradient. Compared with the usual Sephadex G-100 chromatography, HPLC gave a much better separation, with a higher yield and a considerably reduced analysis time (30 min vs 5 h). The [125I]-labelled preparation had normal binding to IM-9 lymphocyte receptors. The maximum bindability of the HPLC-purified preparation approximated 90%, which is the best value so far reported for human growth hormone. It is concluded that HPLC is a fast, convenient and reproducible method for obtaining an improved [125I]-labelled human growth hormone for receptor studies.     

Purification and characterization of luteinizing hormone-releasing hormone from codfish brain

We have purified luteinizing hormone-releasing hormone (LH-RH) from codfish brain and have demonstrated its identity with salmon LH-RH (sLH-RH). An antiserum raised against sLH-RH was used in a specific radioimmunoassay (RIA) to monitor purification and to manufacture an immunoaffinity chromatography column for the initial purification step. The cross-reactivity of the sLH-RH RIA with mammalian LH-RH was 0.1%. Acid extracts of codfish brains were sequentially purified by immunoaffinity chromatography, gel-filtration chromatography, and three steps of reverse-phase HPLC. The purified material and synthetic sLH-RH coeluted on reverse-phase HPLC and exhibited similar biological activity in a dispersed pituitary cell bioassay. Furthermore, the amino acid composition of the purified material was identical to salmon LH-RH. These results suggest that there is structural conservation of LH-RH between these species of teleost fish.     

Characterization and partial purification of human epithelial transforming growth factor

A polypeptide growth factor has been partially purified from medium conditioned by the human adrenocortical carcinoma cell line SW13. This factor, designated h-TGFe, stimulates anchorage-independent growth of the SW13 cells. Similar activity was observed in human milk, and in conditioned media from seven of 14 epithelial cell lines. The SW13-derived activity is stable to low pH and 8M urea but labile to dithiothreitol and 2% sodium dodecyl sulfate. Human TGFe does not bind to heparin and fails to stimulate growth of endothelial cells in monolayer culture. The apparent molecular weight of h-TGFe is 59k by size exclusion chromatography in the presence of 8M urea and the activity binds strongly to cation exchangers. The activity elutes at 15-30% acetonitrile from a C18 reverse-phase column and has been partially purified by using a four-step chromatographic procedure. TGFe appears to be a novel growth factor produced by many epithelial cells and tissues.     

Purification and characterization of human T-lymphocyte-derived erythroid-potentiating activity

The human T-lymphoblast cell line, Mo, secretes a number of lymphokines, including erythroid-potentiating activity (EPA), an important early regulator of erythropoiesis. We report purification of EPA to homogeneity, from serum-free Mo-conditioned medium. Purification was accomplished by sequential concentration, ammonium sulfate precipitation, lentil lectin affinity chromatography, gel filtration, and reverse-phase high-performance liquid chromatography. EPA was assayed by its ability to stimulate the growth of large erythroid colonies (bursts) from normal human peripheral blood. The purified EPA has a molecular weight of 28,000 and appears as a single band when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or nonreducing conditions. Purified EPA stimulates the growth of both early and late erythroid precursors from human bone marrow, as well as colony formation by the K562 human erythroleukemia cell line. Purified EPA has no colony-stimulating factor activity nor does it appear to be a structural protein of the human T-cell leukemia virus subtype II which infects the Mo cells.     

A 60-kDa phosphorylated protein from fetal human bone

A phosphorylated protein was isolated and purified from fetal human bone. Fetal and adult human bones were decalcified with EDTA, and the extract from the fetal bone was fractionated using Q-Sepharose anion exchange chromatography. The fraction containing Ser(P) was purified by Sephacryl S-200 molecular sieving and C4 reverse-phase HPLC. The purified protein had a molecular weight of 60000 on SDS-PAGE, where the protein was stained with Rhodamine-B. The amino acid composition of this protein was different from any other reported phosphorylated proteins in human bone. However, this phosphorylated protein was difficult to detect in the adult bone extract on SDS-PAGE.     

Towards large-scale purification of natural CSF-1 from human placenta tissue extracts

The monocyte-macrophage colony-stimulating factor (colony-stimulating factor 1) is characterized and partially purified from industrially processed human tissues for the first time. A five-step purification procedure using placenta tissue extracts furnished a 13,620-fold enrichment of biological activity. This procedure includes a "pilot" scale anion-exchange chromatography at pH 4.5, gel permeation, and lectin affinity separation followed by HPLC steps (hydrophobic interaction and C18 reverse-phase chromatographies). The purified bioactive material, which stimulates only monocyte-macrophage progenitors and mature cells, showed an Mr of 58,000-62,000 (gel filtration) and an isoelectric point of 3.8-4.0. The hydrophobicity of the molecule was low, and the biological activity was eluted at 50% acetonitrile on a C18 reverse-phase HPLC column. It was totally inactivated by 2-beta-mercaptoethanol reduction and heat treatment. Immunoprecipitation and neutralization of biological activity with specific anti-CSF-1 antibodies (not shown) demonstrated that this material was CSF-1. Step 5 of this protocol yielded two silver-stained bands on 12.5% SDS-PAGE: a major 55-kDa band (96%) and a minor 33-kDa band (4%). CSF-1 was detected exclusively in a band of 52-62 kDa by both Western immunoblotting and bioassays. Immunoaffinity techniques using antibodies directed against selective epitopes on the placental CSF-1 are now considered to purify this material to homogeneity. This approach to the mass production of natural CSF-1 from human tissue has advantages with respect to both the difficulty of post-translational processing of bioactive material in procaryotes and the cost of eucaryotic cell cultures.     

Purification and Characterization of a Cryoprotective Protein (Cryoprotectin) from the Leaves of Cold-Acclimated Cabbage

We have purified a protein (cryoprotectin) from the leaves of cold-acclimated cabbage (Brassica oleracea L.) that protects thylakoids from nonacclimated spinach (Spinacia oleracea L.) against freeze-thaw damage. The procedure involves precipitations by heat, ammonium sulfate, and the glycosaminoglycan heparin and column chromatography on Polyamide 6 and a C18 reverse-phase matrix. After reverse-phase chromatography we obtained a single band of an apparent molecular mass of 7 kD when fractions that showed cryoprotective activity were analyzed by sodium dodecyl sulfate gel electrophoresis and silver staining. Gel-filtration experiments confirmed that the active protein is a monomer of 7 kD native molecular mass. This 7-kD protein could be purified only from cold-acclimated cabbage, but not from plants grown under nonacclimating conditions. Using peroxidase-labeled lectins, we show that cryoprotectin is a glycoprotein and that the saccharide moiety contains [alpha]1-3-linked fucose.     

Interleukin 1 activity in normal human urine

Human leukocyte dialysates contain components capable of amplifying cutaneous delayed-type hypersensitivity (DTH) reactions. In the present study, two such amplifiers, both less than 3500 m.w., were partially purified from human leukocyte dialysates by gel filtration on Sephadex G-10 followed by high pressure reverse-phase liquid chromatography. These amplifiers of DTH were examined for their effects on production of the migration inhibitory lymphokines leukocyte migration inhibition factor (LIF) and macrophage migration inhibition factor (MIF). The amplifiers were found to increase LIF and MIF production by antigen- or alloantigen-stimulated human peripheral blood lymphocytes in a dose-dependent fashion. Further analysis demonstrated that although antigen-stimulated T4 and T8 cell subpopulations could produce LIF activity under the assay conditions employed, amplification of lymphokine production by modulator was only observed with the T4 subset.     

Purification of an embryotrophic factor from commercial bovine serum albumin and its identification as citrate.

A factor of low M(r) with growth-promoting effects on rabbit embryos was extracted and purified from commercial bovine serum albumin (BSA). This embryotrophic factor was extracted from BSA dissolved in formic acid by membrane filtration (membrane cutoff of M(r) 10,000) and then freeze-drying of the filtrate. The extract was purified successively by chromatography on G-10 Sephadex, QAE-Sephadex A-25 anion exchange and high-performance liquid chromatography (HPLC) reverse-phase columns. Mass spectrometry of the active reverse-phase material indicated that the major component in this material had an M(r) of 192. The embryotrophic factor in the low M(r) extract of BSA was shown to be citrate, because: (i) the mass spectra of the active reverse-phase material and citrate were identical, (ii) the activity was eluted at the identical position to citrate on an analytical HPLC anion-exchange column, (iii) the original BSA sample was shown by enzyme assay to be heavily contaminated by citrate and (iv) citrate stimulated cell proliferation and expansion of blastocysts.     

High-level expression and purification of human epidermal growth factor with SUMO fusion in Escherichia coli

Human epidermal growth factor (hEGF) can stimulate the division of various cell types and has potential clinical applications. However, the high expression of active hEGF in Escherichia coli has not been successful, as the protein contains three intra-molecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the hEGF gene with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO-hEGF fusion gene that was highly expressed in Origami (DE3) strain. The optimal expression level of the soluble fusion protein, SUMO-hEGF, was up to 38.9% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease to obtain the native hEGF, which was further purified by Ni-NTA affinity chromatography. The result of the reverse-phase HPLC showed that the purity of the recombinant cleaved hEGF was greater than 98%. The primary structure of the purified hEGF was confirmed by N-terminal amino acid sequencing and MALDI-TOF mass spectroscopy analysis. Using the method of methylthiazoletetrazolium, the mitogenic activity on Balb/c 3T3 cells of the purified hEGF was comparable to that of commercial hEGF.     

Identification of [hydroxyproline3]-lysyl-bradykinin released from human kininogens by human urinary kallikrein

The types of kinins released from purified native, single chain human high and low molecular mass kininogens (HMMKs and LMMKs, respectively) by purified human urinary kallikrein were separated by reverse-phase HPLC and quantitated by the rat uterus bioassay. [Hyp3]-lysyl-bradykinin, a recently discovered kinin, represented up to 58% of the biological activity released from 4 individual HMMK preparations purified from 4 different healthy volunteers. In contrast, the majority of the biological activity released from LMMKs purified from pooled plasma was identified as Lys-bradykinin and [Hyp3]-lysyl-bradykinin represented only 6.4 +/- 3.8%. These findings indicate posttranslation hydroxylation of human kininogens and suggest a preference of HMMKs for this modification.     

Purification and MALDI-MS characterization of stressin, a stress-associated glycoprotein.

Glycoconjugates have a whole spectrum of biological roles, from those that appear trivial to those that are crucial. Results accumulated in the past years indicate they might also play an important role in the response to stress, a complex physiological response of the human organism to various threats. We have recently identified stressin, a human serum glycoprotein, which was found to be increased under stress conditions. Here we report the purification of stressin from sera of professional soldiers and partial characterization of its protein and carbohydrate parts using lectin blotting and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Stressin was purified using a combination of ammonium sulfate precipitation, ion exchange chromatography, preparative gel electrophoresis and reverse-phase HPLC. It was found to be a highly glycosylated protein. Only 21.9 kDa (out of 36.7 kDa) was the protein part, whereas the remaining 40% of the mass originated from N-linked oligosaccharides. The carbohydrate part contained 12 sialic acids moieties, nearly 90% of which were lost due to post-source decay in the field-free tube. Tryptic fragments were produced from glycosylated and deglycosylated stressin, separated by reverse-phase HPLC and their exact molecular masses were determined using MALDI-MS. Comparison with tryptic maps of other proteins in computer databases indicated that stressin does not correspond to any already described protein.     

Production and purification of refolded recombinant human IL-7 from inclusion bodies

A recombinant form of human rhIL-7 was overexpressed in Escherichia coli HMS174 (DE3) pLysS under the control of a T7 promoter. The resulting insoluble inclusion bodies were separated from cellular debris by cross-flow filtration and solubilized by homogenization with 6 M guanidine HCl. Attempts at refolding rhIL-7 from solubilized inclusion bodies without prior purification of monomeric, denatured rhIL-7 were not successful. Denatured, monomeric rhIL-7 was therefore initially purified by size-exclusion chromatography using Prep-Grade Pharmacia Superdex 200. Correctly folded rhIL-7 monomer was generated by statically refolding the denatured protein at a final protein concentration of 80-100 microg/ml in 100 mM Tris, 2mM EDTA, 500 mM L-arginine, pH 9.0, buffer with 0.55 g/l oxidized glutathione at 2-8 degrees C for at least 48 h. The refolded rhIL-7 was subsequently purified by low-pressure liquid chromatography, using a combination of hydrophobic interaction, cation-exchange, and size-exclusion chromatography. The purified final product was >95% pure by SDS-PAGE stained with Coomassie brilliant blue, high-pressure size-exclusion chromatography (SEC-HPLC), and reverse-phase HPLC. The endotoxin level was <0.05 EU/mg. The final purified product was biologically active in a validated IL-7 dependent pre-B-cell bioassay. In anticipation of human clinical trials, this material is currently being evaluated for safety and efficacy in non-human primate toxicology studies.     

Identification of a 15,000-molecular-weight form of immunoreactive transforming growth factor alpha in extracts of porcine pituitary

Two different mitogenic activities were identified from extracts of porcine pituitary by using COMMA-D mouse mammary epithelial cells in a serum-free 3H-thymidine incorporation assay. Porcine pituitaries were extracted in phosphate-buffered saline (pH 7.4) and 25-80% (NH4)2SO4 pellets were dialyzed and chromatographed by using DEAE-Sepharose chromatography (pH 8.0), resulting in two peaks (I and II) of mitogenic activity. Peak I represented a recovery of 73% of the units of mitogenic activity present in crude extract of pituitary while only 1.25% of the activity was recovered in peak II. Peak I was further purified by using CM-Sephadex and heparin-Sepharose chromatographies and yielded a mitogen that was able to elicit one-half-maximal stimulation of 3H-thymidine incorporation by COMMA-D cells at 48 pg/ml. As expected with pituitary as the tissue source, peak I was confirmed to be basic fibroblast growth factor (bFGF) by using specific antibodies in enzyme-linked immunosorbent assay and Western immunoblotting procedures. Peak II was further purified by using chromatofocusing (pH 7.3-5.0), reverse-phase, and cation-exchange HPLCs. The mitogenic activity eluted at pH 6.3 from chromatofocusing, migrated as a 13-kDa molecule on gel filtration HPLC, and did not bind to heparin-Sepharose under conditions which bound fibroblast growth factors. The material purified from peak II and rat synthetic transforming growth factor alpha (TGF alpha) competed in a parallel fashion with 125I-epidermal growth factor for receptor binding with A431 human epidermal carcinoma cells. In addition, the mitogen purified from peak II showed a single immunoreactive band migrating at 15 kDa when specific antiserum against TGF alpha was used in a Western immunoblotting procedure. The data suggest that in addition to the well-documented presence of bFGF, normal adult porcine pituitaries contain a 15-kDa form of immunoreactive TGF alpha that binds to EGF receptors and is mitogenic for mammary epithelial cells.     

Recombinant human erythropoietin: purification and analysis of carbohydrate linkage

Erythropoietin was purified to homogeneity from the culture medium of a baby hamster kidney cell line stably transfected with a human erythropoietin gene. A three-step procedure was used, which included affinity chromatography, ion-exchange chromatography, and reverse-phase chromatography. Purity of the protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequence analysis. Overall recovery was 35%. The biological activity of purified recombinant erythropoietin was similar to that of the native hormone in vitro. The purified recombinant hormone contained N-linked carbohydrate at residues 24, 38, and 83, and and O-linked carbohydrate at residue 126.     

Characterization and NH2-terminal amino acid sequence of natural human interleukin for DA cells: leukemia inhibitory factor. Differentiation inhibitory activity secreted by a T lymphoma cell line

Six human T lymphomas and an NK-like cell line were tested for their ability to produce HILDA, one of the two human growth promoting activities for the DA1.a cells. Among them, the HSB2 cell line turned out to be the only one secreting significant HILDA activity (200-400 units/ml) after activation with 50 nM phorbol myristate acetate. Subclones of the HSB2 cell line were obtained by limiting dilution experiments. One of them (2B3) was found to secrete 1,000-5,000 units/ml of HILDA after phorbol myristate acetate activation in the presence of 10% fetal calf serum and 200-500 units/ml in serum-free conditions. 2B3-HILDA was purified from serum-free conditioned medium by a four-step procedure including fast flow cationic exchange at pH 6, concanavalin A chromatography, reverse-phase high performance liquid chromatography and gel-filtration high performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified radiolabeled cytokine revealed a single band of Mr 43,000, which co-electrophoresed with the biological activity. The NH2-terminal amino acid sequence of the first residues of the protein were determined and found to be similar to the equivalent residues deduced from the molecularly cloned cytokine. Isoelectric point determination revealed some charge heterogeneity of HILDA, which focused to pH 8.5-9 after neuraminidase treatment. Carbohydrate content of the cytokine was studied by deglycosylation experiments which showed that the O-linked oligosaccharides represented 2,000-3,000 and that the N-linked sugars account for half of the apparent molecular weight of HILDA.