Recombinant human glucagon: Large-scale purification and biochemical characterization

Recombinant glucagon was expressed inEscherichia coli as a fusion protein including the glucagon sequence therein as previously reported [Ishizakiet al. (1992).Appl. Microbiol. Biotechnol.36, 483–486]. We developed a large-scale method for the isolation and purification of recombinant glucagon. After cell disruption, the resultant pellets were solubilized with 2 M guanidine-HCl, to whichStaphylococcus aureus V8 protease had been added, and were digested into intermediates composed of 53- and 60-residue peptides containing the glucagon moiety. After the digestion came to an end, the solution was desalted, and the remaining V8 protease was allowed to resume digestion of the intermediates into glucagon, followed by partial purification by S-Sepharose and Sephacryl S-100 chromatographies. The glucagon obtained was found to be not less than 99.5% pure by analytical HPLC. One liter of culture produced about 180 mg of pure glucagon. The amino acid composition and the sequence agreed well with the theoretical values. Radioreceptor assay gave an affinity constant similar to that of pancreatic glucagon, and similar activities in cAMP production and glycogenolysis were also observed. Thus, the recombinant glucagon was confirmed to be biochemically identical with pancreatic glucagon.     

Molecular cloning, overexpression and characterization of human interleukin 1alpha

Interleukin-1 alpha (IL-1alpha) regulates a wide range of important cellular processes. In this study for the first time, we report the cloning, expression, biophysical, and biological characterization of the human interleukin-1alpha. Human IL-1alpha has been expressed in Escherichia coli in high yields ( approximately 4mg per liter of the bacterial culture). The protein was purified to homogeneity ( approximately 98% purity) using affinity chromatography and size exclusion chromatography. Results of the steady-state fluorescence and 2D NMR experiments show that the recombinant IL-1alpha is in a folded conformation. Far-UV circular dichroism (CD) data suggest that IL-1alpha is an all beta-sheet protein with a beta-barrel architecture. Isothermal titration calorimetry (ITC) experiments show that the recombinant IL-1alpha binds strongly (K(d) approximately 5.6 x 10(-7) M) to S100A13, a calcium binding protein that chaperones the in vivo release of IL-1alpha into the extracellular compartment. Recombinant IL-1alpha was observed to exhibit strong cytostatic effect on human umbilical vascular endothelial cells. The findings of the present study not only pave way for an in-depth structural investigation of the molecular mechanism(s) underlying the non-classical release of IL-1alpha but also provide avenues for the rational design of potent inhibitors against IL-1alpha mediated pathogenesis.     

Preparation and characterization of polyclonal and monoclonal antibodies against human interleukin 1 alpha (IL 1 alpha)

Polyclonal and monoclonal anti-human IL 1 alpha antibodies (Ab) have been established. These Ab neutralized human recombinant IL 1 alpha (rIL 1 alpha) activity effectively, but did not interfere with human rIL 1 beta, murine rIL 1 alpha, or human rIL 2 activity. Fifty percent of rIL 1 alpha activity (25 U/ml, or 2.5 ng/ml) was neutralized by less than 0.06 microgram/ml of rabbit anti-IL 1 alpha Ab (R-38.3G) and by less than 0.13 microgram/ml of monoclonal Ab (clone 28(3B1], respectively. In other experiments, 10 micrograms/ml of rabbit anti-IL 1 alpha Ab could effectively neutralize 50% of 2000 U of rIL 1 alpha activity, and the same amount of monoclonal Ab neutralized 50% of 500 U/ml of rIL 1 alpha activity. Not only IL 1 alpha activity in the thymocyte costimulator assay, but also IL 1-dependent IL 2 production by a human leukemic cell line, HSB.2 subclone, were blocked by these polyclonal or monoclonal Ab. In addition, pI 4.9 IL 1 activity produced by the myelomonocytic cell line THP-1 and by the Epstein-Barr virus-transformed B cell lines, were neutralized by these Ab, suggesting that these cell lines also produce IL 1 alpha. The specificity of these polyclonal and monoclonal Ab was further confirmed by immunochemical method (Western blotting), in which anti-IL 1 alpha Ab reacted with rIL 1 alpha in a specific manner. Furthermore, an enzyme-linked immunosorbent assay system has been developed that can detect low levels of IL 1 alpha activity (less than 0.3 ng/ml or less than 3 U/ml), which is still less sensitive than thymocyte comitogenic assay and considerably less sensitive than the D10 assay. Finally, anti-IL 1 alpha Ab-conjugated affinity columns were prepared, by which IL 1 alpha activity, but not IL 1 beta activity, was specifically adsorbed and eluted effectively.     

Interleukin 1 induces interleukin 1. II. Recombinant human interleukin 1 induces interleukin 1 production by adult human vascular endothelial cells

Interleukin 1 (IL-1) alters several potentially pathogenic endothelial cell (EC) functions. The authors report here that recombinant human IL-1 (rIL-1) alpha (0.1 to 10 ng/ml) or IL-1-beta (1 to 100 ng/ml) induce concentration- and time-dependent increases in IL-1-beta mRNA levels in EC derived from adult human saphenous vein. rIL-1 induced IL-1-alpha mRNA only in EC treated concomitantly with cycloheximide (2 micrograms/ml). IL-1-beta mRNA production began within 1 hr of exposure to rIL-1, peaked after 24 hr, and declined thereafter. Actinomycin D prevented the appearance of IL-1 mRNA in rIL-1-treated EC. rIL-1 also induced the release of biologically active IL-1 from EC, which was inhibited by cycloheximide (1 microgram/ml). When compared on the basis of their activity in the thymocyte costimulation assay, rIL-1-alpha and rIL-1-beta were equipotent as inducers of IL-1 production by EC. EC stimulated with rIL-1 produced prostaglandin E2, which inhibits IL-1 production by other cell types and also decreases the responsiveness of thymocytes to IL-1. When EC were exposed to rIL-1 in the presence of indomethacin (1 microgram/ml), which blocked prostaglandin E2 production, greater amounts of rIL-1-induced IL-1 release were detected, although the inhibitor did not affect IL-1-beta mRNA levels. IL-1-induced IL-1 production was unlikely to be caused by endotoxin contamination of tissue culture media or IL-1 preparations, because the lipopolysaccharide (LPS) antagonist polymyxin B (10 micrograms/ml) blocked LPS-induced IL-1 production by EC but did not affect IL-1 release in response to rIL-1-beta (100 ng/ml). The IL-1-inducing property of rIL-1-beta was heat-labile, whereas heated LPS stimulated EC IL-1 production. The source of IL-1 in our cultures was not monocyte/macrophages, as treatment of EC with monoclonal antibody to the monocyte antigen Mo2 under conditions that lysed adherent peripheral blood monocytes did not affect production of IL-1 by EC in response to LPS (1 microgram/ml) or rIL-1-beta (100 ng/ml). IL-1 elicits a coordinated program of altered endothelial function that increases adhesiveness for leukocytes and coagulability. IL-1-induced IL-1 gene expression in human adult EC could thus provide a positive feedback mechanism in the pathogenesis of vascular disease including atherosclerosis, vasculitis, and allograft rejection.     

Human interleukin 1: purification and properties

Human interleukin 1 (IL 1) has been purified to homogeneity by a procedure of molecular weight fractionation, isoelectric focusing, and preparative polyacrylamide gel electrophoresis. The homogeneity of the purified material has been demonstrated by silver staining of analytical polyacrylamide gels. The homogeneous IL 1 retains only a trace of its original biological activity because of the denturing effects of the sodium dodecyl sulfate used in the final step of purification. Very highly purified IL 1, retaining strong biological activity, has been eluted from nondenaturing polyacrylamide gels. This IL 1 has been demonstrated to stimulate human and mouse T and B lymphocytes, fibroblasts, and synovial cells. In addition, in vivo treatment of animals with IL 1 resulted in the immunologically relevant symptoms of fever, increased plasma levels of acute phase proteins, and increased numbers of circulating neutrophils.     

Production, purification, and characterization of human alpha1 proteinase inhibitor from Aspergillus niger

Human alpha one proteinase inhibitor (alpha1-PI) was cloned and expressed in Aspergillus niger, filamentious fungus that can grow in defined media and can perform glycosylation. Submerged culture conditions were established using starch as carbon source, 30% dissolved oxygen concentration, pH 7.0 and 28 degrees C. Eight milligrams per liter of active alpha1-PI were secreted to the growth media in about 40 h. Controlling the protein proteolysis was found to be an important factor in the production. The effects of various carbon sources, pH and temperature on the production and stability of the protein were tested and the product was purified and characterized. Two molecular weights variants of the recombinant alpha1-PI were produced by the fungus; the difference is attributed to the glycosylated part of the molecule. The two glycoproteins were treated with PNGAse F and the released glycans were analyzed by HPAEC, MALDI/TOF-MS, NSI-MS(n), and GC-MS. The MALDI and NSI- full MS spectra of permethylated N-glycans revealed that the N-glycans of both variants contain a series of high-mannose type glycans with 5-20 hexose units. Monosaccharide analysis showed that these were composed of N-acetylglucos-amine, mannose, and galactose. Linkage analysis revealed that the galactosyl component was in the furanoic conformation, which was attaching in a terminal non-reducing position. The Galactofuranose-containing high-mannnose type N-glycans are typical structures, which recently have been found as part of several glycoproteins produced by Aspergillus niger.     

Chromosomal localization of the human interleukin 1 alpha (IL-1 alpha) gene

The human interleukin 1 alpha gene was assigned to chromosome 2 using Southern transfer analysis of human-rodent somatic cell hybrid DNAs. The gene was regionally localized to 2q12-21 using in situ hybridization to metaphase chromosomes. These results indicate that the IL-1 alpha gene maps to the same general region on the long arm of chromosome 2 as the IL-1 beta gene, which has been previously assigned.     

Receptor-mediated endocytosis and nuclear transport of human interleukin 1 alpha.

In this study we demonstrate that 125I-labelled interleukin (IL) 1 alpha binds specifically to its receptor on the surface of EL4 6.1 cells and is subsequently endocytosed and translocated from the cell membrane to the nucleus, where it progressively accumulates. Two-dimensional polyacrylamide-gel electrophoresis revealed that the internalized 125I-IL1 alpha associated with the nucleus was intact, with negligible breakdown products present. Specific and saturable binding of 125I-IL1 alpha was demonstrated on purified nuclei isolated from these cells. Binding of the radiolabelled ligand showed similar kinetics to that of the plasma-membrane receptor, and was inhibited by both unlabelled IL1 alpha and IL1 beta. Equilibrium binding studies on isolated nuclei revealed a single high-affinity binding site, with a Kd of 17 +/- 2 pM, and 79 +/- 12 binding sites per nucleus. These studies demonstrate that receptor-mediated endocytosis of IL1 results in its accumulation in the nucleus, and this mechanism may play an important role in mediating some of the actions of IL1.     

Expression, purification, and characterization of recombinant human interleukin 24 in Escherichia coli

Interleukin-24 (IL-24) can induce apoptosis of a broad range of tumor cells, and this function of IL-24 is independent of classic tumor suppressor genes, such as p53, Rb and p16. Here, we report the expression, purification and preparation of a recombinant IL-24 protein (rIL-24) without post-translational modifications, which may selectively induce apoptosis of tumor cells in vitro. We found that non-fusion rIL-24 was not able to be expressed by vectors pET11c, 28a, and 22b in Escherichia coli. To obtain recombinant non-fusion IL-24 protein, the encoding region for IL-24 was cloned between KpnI and BamHI in pET32a. The Trx (Thioredoxin)/IL-24 fusion proteins were expressed in the form of inclusion bodies in E. coli host strain BL21 (DE21). The expression level was more than 30% of total cell lysate. Inclusion bodies were disrupted, washed, and isolated at pH 9.0, and were completely dissolved in a buffer containing 2M urea at pH 9.0. After nickel ion metal affinity chromatography, gel filtration chromatography, and renaturation, the refolded fusion proteins with a purity of >96% were obtained. Trx/IL-24 proteins were digested by enterokinase (EK) to both Trx and rIL-24 fragments which then were separated by cation exchange chromatography. Cell proliferation experiments proved that the rIL-24 (98% purity) retains its cancer-selective apoptosis-inducing properties. This result suggested that the rIL-24 may have cancer therapeutic applications.     

Expression and purification of recombinant human CCL5 and its biological characterization

The Protein Journal - C-C motif chemokine ligand 5 (CCL5) is crucial in the tumor microenvironment. It has been previously reported to act as a key role in tumor invasion and metastasis. However,...     

Cloning, expression, purification, and biological activity of recombinant native and variant human alpha 1-antichymotrypsins

Human alpha 1-antichymotrypsin has been cloned, sequenced and expressed in Escherichia coli and recombinant protein as well as point-specific mutants have been purified and characterized. The corrected gene-deduced amino acid sequence has 45% overall identity with alpha 1-protease inhibitor, which is higher than the 42% previously reported (Chandra, T., Stackhouse, R., Kidd, V. J., Robson, J. H., and Woo, S. L. C. (1983) Biochemistry 22, 5055-5060). Recombinant antichymotrypsin (rACT) is similar to natural antichymotrypsin with respect to the specificity of its interactions with proteases. Its second-order rate constant for association with bovine chymotrypsin is 6-8 x 10(5) M-1 s-1, which is identical to that of the serum-derived inhibitor. Site-specific mutagenesis has been used to produce two variants of rACT in which the P1 position has been changed from leucine to either methionine (L358M-rACT) or arginine (L358R-rACT). L358M-rACT has a specificity of inhibitory activity toward serine proteases closely similar to that of native rACT. By contrast, the specificity of L358R-rACT is quite different from that of native rACT, most notably in efficiently inhibiting trypsin and human thrombin while showing a decreased ability to inhibit chymotrypsin.     

Purification and characterization of human recombinant precursor interleukin 1 beta

We have purified the 31-kDa precursor of human interleukin 1 beta (proIL1 beta) from recombinant Escherichia coli expressing the protein. The recombinant precursor was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, Western blot, and for biological and receptor binding activity. The protein migrates at the expected molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration columns. The specific activity of the recombinant precursor is less than 10(2) units/mg in the EL4 thymoma assay compared with 5 x 10(8) units/mg for the recombinant 17-kDa mature protein. The inactivity of the precursor is attributable to the inability of the protein to bind the IL1 receptor on EL4 cells as shown by receptor competition studies using 125I-labeled 17-kDa IL1 beta. Inactivity of the IL1 beta precursor is not due to degradation of the protein in either the bioactivity or receptor binding assays. The inactive IL1 beta precursor is converted to an active form following proteolysis with chymotrypsin which generates a carboxyl-terminal fragment of 17 kDa that is 6 orders of magnitude more active than the starting IL1 beta precursor. Removal of the first 114 amino acids from proIL1 beta generates a fully active molecule. In contrast, removal of the first 77 amino acids by treatment with trypsin only partially restores activity. The resultant 22-kDa protein exhibits a 600-fold increase in both biological and receptor binding activity, demonstrating a direct correlation between the ability of sequences within the pro-region to inhibit biological activity and inhibit binding to the IL1 receptor. Far-UV circular dichroism spectroscopy indicates that proIL1 beta is similar in secondary structure to mature IL1 beta; both proteins are nonhelical beta sheet proteins.     

Recombinant thymosin alpha1

An artificial gene encoding thymosin alpha1 was obtained by the chemoenzymatic synthesis and cloned into Escherichia coli. An expressing recombinant plasmid containing the hybrid protein gene, which encodes amino acid sequences of thymosin alpha1 and the Saccharomyces cerevisiae intein Sce VMA, was constructed. The expression of the hybrid protein from the resulting hybrid gene in E. coli, the properties of the resulting hybrid protein, and the conditions for its nonenzymatic cleavage to thymosin alpha1 were studied. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.     

Recombinant expression, affinity purification and functional characterization of Scots pine defensin 1

Plants produce a variety of molecules to defend themselves from fungal pathogens. Defensins belong to the family of antimicrobial peptides that play a central role in innate immunity in all species of plants. We have previously reported the purification of antimicrobial peptides from Scots pine seedlings and the identification of some of them, including defensin, by mass spectrometry. In this study, we extend our original study on molecular cloning of Pinus sylvestris defensin 1 (PsDef1) by presenting the expression and affinity purification of recombinant defensin 1 (rPsDef1). The full-length coding sequence of PsDef1 has an open reading frame capable to encode a protein of 83 amino residues, including a signal peptide of 33 aa, followed by a characteristic defensin domain of 50 amino acids representing its active form. The calculated molecular weight of the mature form of PsDef1 is 5,601.6 Da. We have employed pET system to express mature form of PsDef1 fussed to GST. As GST-PsDef1 fusion protein was not biologically active, we removed GST moiety from the mature defensin 1 peptide by proteolytic cleavage with Factor Xa. The resulting rPsDef1 protein exhibited strong antifungal activity against a panel of pathogenic fungi which is comparable to that of endogenous Scots pine defensin 1. In addition, rPsDef1 was used to produce specific polyclonal antibodies. Using generated antibodies, we found that the level of PsDef1 is significantly increased in Scots pine seedlings during germination and in their response to pathogenic infection with Heterobasidion annosum.     

High-efficiency purification and chemical characterization of B cell stimulatory factor-1/interleukin 4

B cell stimulatory factor-1/interleukin 4, a lymphokine produced by phorbol ester activated-EL-4 thymoma cells, was purified to homogeneity in good yield by a two-step purification procedure, using affinity chromatography and a single subsequent round of reverse-phase high-performance liquid chromatography. The N-terminal sequence of the first 24 amino acids was consistent with that inferred from the nucleotide sequence of BSF-1 cDNA clones. Amino acid composition analysis also agreed well with that predicted from the nucleotide sequence. A rabbit antibody to a peptide corresponding to positions 100 to 113 inferred from the nucleotide sequence of the cDNA clones bound to BSF-1 purified from EL-4 cells. Purified BSF-1 possesses complex N-linked glycosidic side chains as shown by reduction in Mr from 20,000 to approximately 15,000 by endoglycosidase F but not by endoglycosidase H treatment. Removal of these N-linked sugars does not diminish the activity of BSF-1 as a costimulant in the response of B cells to anti-IgM. By contrast, the reduction of disulfide bonds completely destroyed biologic activity. A monoclonal antibody to BSF-1 blocks its binding to cellular receptors and inhibits biological activities, whereas antibody to the BSF-1 peptide (100-113) has neither effect.     

A three-step purification of human alpha 1-acid glycoprotein

alpha 1-Acid glycoprotein (AGP) was purified to homogeneity by a 3-step procedure using pseudo-ligand affinity chromatography on immobilized Cibacron blue F3GA, Procion red HE3B, and preparative column isoelectric focusing. The overall yield of the combined techniques was 88%. Analysis of the purified AGP by lectin affinity chromatography on immobilized Con A and immunoaffino-electrophoresis indicated that the most acidic form did not interact with the lectin, while the two more basic fractions possessed different affinities for Con A. In addition, 3 different populations of AGP were clearly separated by Con A affinity chromatography.     

Affinity purification and characterization of recombinant human galectin-1

Galectin-1, a polypeptidic factor that can have major effects on cell growth and apoptosis, was overexpressed in E. coli. This protein was purified to homogeneity by affinity chromatography on lactose coupled to divinylsulfone-activated agarose. The recombinant galectin-1 (rGAL1) was compared with the homologous protein purified from human brain tissue using two-dimensional electrophoresis on immobilized pH gradient (IPG-DALT). rGAL1 had a major isoelectric point of 5.4 (major pI of tissular galectin-1, 5.1) and its subunit molecular mass was 14 500. Addition of rGAL1 to Jurkat T-lymphoblastoid cells induced cell death in a concentration-dependent manner.     

Calcium-dependent binding of phosphorylated human pre interleukin 1 alpha to phospholipids

The effect of phosphorylation of pre interleukin 1 alpha (IL 1 alpha) on its association with various phospholipids was investigated. We prepared genetically engineered truncated human pre IL 1 alpha (residues 64 to 271) and phosphorylated this pre IL 1 alpha in vitro by using the catalytic subunit of cAMP-dependent protein kinase. Phosphorylated truncated pre IL 1 alpha selectively binds to acidic phospholipids including phosphatidic acid, phosphatidylserine, and phosphatidylinositol, but not to other phospholipids (phosphatidylcholine and phosphatidylethanolamine). This binding required divalent cations: Ca2+ or Mn2+, but not Mg2+. In order to obtain half-maximal binding of pre IL 1 alpha to phosphatidic acid or phosphatidylserine, Ca2+ between 5 and 100 microM was required. Unphosphorylated pre IL 1 alpha did not bind to phosphatidylserine, indicating that phosphorylation is required for this binding. Phosphorylated pre IL 1 alpha did not bind to intact peripheral blood mononuclear cells irrespective of lipopolysaccharide stimulation, but did bind to membrane vesicles prepared from these cells in the presence of calcium. Furthermore, phosphorylated pre IL 1 alpha bound only to inside-out ghosts, but not right-side-out ghosts, prepared from human red blood cells. Taken together, these data suggest that phosphorylated pre IL 1 alpha binds to the inner surface of plasma membrane in a Ca2(+)- and phospholipid-dependent manner.     

Recombinant human prorenin from CHO cells: Expression and purification

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from 1–5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence andpH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P₆-P₃ sequence of human angiotensinogen.     

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.