Production, purification, and characterization of recombinant prohormone convertase 5 from baculovirus-infected insect cells

The discovery of the prohormone convertase (PC) family of enzymes has provided several good candidates (PC1, PC2, and PC5) for the enzymes responsible for the endoproteolytic cleavage of procholecystokinin (pro-CCK). Determination of the role of individual pro-hormone convertases in the processing of pro-CCK is complicated because several of these enzymes are found in endocrine tumor cells expressing CCK mRNA and in identified neurons in the brain. Production of active recombinant PC5 permits the determination of its ability to cleave substrates related to pro-CCK. Active PC5, secreted from baculovirus-infected Sf9 cells, was partially purified by ion-exchange chromatography. Western blot analysis confirmed the presence of the active form of the enzyme in infected cell media and its absence from uninfected cell media. The enzyme is most active at acidic pH 6.5 and is maximally activated by 5 mM calcium. PC5 was able to cleave both monobasic and dibasic substrates without a requirement for a basic residue at P-4 and it displayed a K(m) in the micromolar range. The enzyme was inhibited by EDTA, 1,10-phenanthroline, and p-CMS, as well as by two specific PC inhibitors. This is the first reported preparation of active recombinant PC5. Like the other members of its family, it has the correct catalytic characteristics in vitro to play a role in the processing of neuropeptide precursor proteins into their final bioactive forms.     

Production of useful recombinant proteins using Namalwa KJM-1 cells adapted to serum-free medium]

We investigated the basic technology of cell culture conditions for production of useful substances such as cytokines, and related proteins produced by Namalwa cells. Namalwa cells, human B lymphoblastoid cells, were used for large scale production of alpha-interferon. Namalwa KJM-1, a subline of Namalwa cells, adapted to serum- and albumin-free medium, can grow at a high density above 1 x 10(7) cells/ml in suspension manner by the use of a perfusion culture system, Biofermenter, containing a cone-type cell-sedimentation column as cell separator. Several kinds of cytokine cDNA can be introduced and expressed in Namalwa KJM-1 cells. Some of these were produced in large quantities by use of a gene amplification method with dhfr, even through the Namalwa KJM-1 cells contained endogenous dhfr genes. For stable production of the target protein, Namalwa KJM-1 cells are very useful host cells, because they have no effective endogenous protease activity in the conditioned medium. Using Biofermenter with micro-silicone fibers and a dialysis system, the specific productivity of the target proteins was not depressed at a high cell density.     

Identification of conditioned medium proteins from human cancer cells adapted to serum-free conditions

Recent studies have shown that human cancer cell lines can be adapted to grow in serum-free, unsupplemented RPMI-1640 (RO) medium. We have developed similar techniques to rapidly identify proteins of interest in serum-free conditioned medium (CM) of human lung cancer cell lines. Classic and variant small cell lung cancer (SCLC) lines were adapted to growth in RO medium. CM from each line was concentrated and fractionated on an anion-exchange column of a fast protein liquid chromatography system. Concentrates of each fraction were loaded onto lanes of minigels of an automated electrophoresis system. Analysis of the chromatograms reveals peaks seen only in CM of the classic SCLC lines. Electrophoretic analysis of the fractions containing these peaks reveal protein bands distinguishing between the subtypes of human SCLC. One protein was purified to homogeneity with subsequent reversed-phase chromatography and identified by protein microsequencing as histone H2B. These automated techniques have general use in the rapid identification of CM proteins associated with the differentiation or progression of the many types of neoplastic cells which can be adapted to growth in RO medium.     

Production, purification, and characterization of alpha-amylase from Thermomonospora curvata.

Thermomonospora curvata produces an extracellular alpha-amylase. Maximal amylase production by cultures in a starch-mineral salts medium occurred at pH 7.5 and 53 degrees C. The crude enzyme was unstable to heating (65 degrees C) at pH 4 to 6, and was activated when heated at pH 8. The enzyme was purified 66-fold with a 9% yield and appeared homogeneous on discontinuous gel electrophoresis. The pH and temperature optima for activity of the purified enzyme were 5.5 to 6.0 and 65 degrees C. The molecular weight was calculated to be 62,000. The Km for starch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture of maltotetraose and maltopentaose.     

Production, purification, and characterization of alpha-amylase from Thermomonospora curvata.

Thermomonospora curvata produces an extracellular alpha-amylase. Maximal amylase production by cultures in a starch-mineral salts medium occurred at pH 7.5 and 53 degrees C. The crude enzyme was unstable to heating (65 degrees C) at pH 4 to 6, and was activated when heated at pH 8. The enzyme was purified 66-fold with a 9% yield and appeared homogeneous on discontinuous gel electrophoresis. The pH and temperature optima for activity of the purified enzyme were 5.5 to 6.0 and 65 degrees C. The molecular weight was calculated to be 62,000. The Km for starch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture of maltotetraose and maltopentaose.     

Production, purification, and characterization of an extracellular chitosanase from Streptomyces.

The synthesis by Streptomyces sp. no. 6 of an extracellular chitosanase was induced by glucosamine. The enzyme was purified to homogeneity by Sephadex G-100, carboxymethyl-cellulose, and diethylaminoethyl-cellulose chromatography. The purified enzyme hydrolyzed chitosan (the beta-1,4-linked polymer of glucosamine) but not chitin nor carboxymethyl-cellulose. The only products of the hydrolysis detectable by paper chromatography were di- and triglucosamine. Sephadex G-100 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the molecular weight of the enzyme was between 29,000 and 26,000. Acid hydrolysates of the enzyme contained no cysteic acid or glucosamine or other carbohydrate. At 25 C, maximum activity was obtained between pH 4.5 and 6.5. The enzymatic hydrolysis of chitosan occurred over a wide range of temperatures and was maximal at 60 C. The rate of the reaction was inhibited by concentrations of soluble chitosan higher than 0.5 g/liter. The apparent Km calculated from a Lineweaver-Burke plot was 0.688 g/liter at pH 5.5. The enzyme prevented spore germination and caused a significant decrease in the turbidity of germinated spore suspensions of the Mucor strains tested. Such a decrease was the result of a partial lysis of the cell wall.     

Production and purification of human menin from Drosophila melanogaster S2 cells using stirred tank reactor

A process was developed for producing human menin from transformed Drosophila Schneider 2 cells. Protein expression was achieved after inducing the metallothionein promoter by adding copper sulfate to cells growing in suspension in a stirred-tank reactor. Experiments in shake flasks showed that the production of menin was improved when the induction was conducted late in the exponential phase of cell growth at a concentration of 1–2 × 10⁷ cells ml^-1, with a copper concentration of 0.2 mM for no more than 24 h. This observation was confirmed by experiments in bench-scale fermentors. Subsequently, a pilot-scale fermentation yielded 1 mg l^-1 culture of purified menin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production, purification, characterization, and applications of lipases

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyze the hydrolysis and the synthesis of esters formed from glycerol and long-chain fatty acids. Lipases occur widely in nature, but only microbial lipases are commercially significant. The many applications of lipases include speciality organic syntheses, hydrolysis of fats and oils, modification of fats, flavor enhancement in food processing, resolution of racemic mixtures, and chemical analyses. This article discusses the production, recovery, and use of microbial lipases. Issues of enzyme kinetics, thermostability, and bioactivity are addressed. Production of recombinant lipases is detailed. Immobilized preparations of lipases are discussed. In view of the increasing understanding of lipases and their many applications in high-value syntheses and as bulk enzymes, these enzymes are having an increasing impact on bioprocessing.     

Pharmacological characterization and immunoaffinity purification of metabotropic glutamate receptor from Drosophila overexpressed in Sf9 cells

Metabotropic glutamate receptors (mGluRs) play important roles in the function and regulation of the central nervous system. Structural studies are necessary for the detailed understanding of their mechanisms of action. However, overexpression and purification of functional receptors in quantities required for these studies proves to be a major challenge. In this study we report the overexpression of a Drosophila melanogaster mGluR (DmGluRA) by using a baculovirus-insect cell expression system. Expression was tested in two different insect cell hosts (Sf9 and Hi5) and analyzed by performing expression kinetics. Pharmacological characterization of the recombinant receptor by radioactive glutamate binding assays showed a profile similar to group II mGluRs, as previously reported, when the receptor was expressed in mammalian systems. The B(max) value reached 11 pM receptor/mg Sf9-membrane protein. A monoclonal antibody against DmGluRA was generated by genetic immunization and used to purify the receptor.     

Drosophila topoisomerase I: isolation, purification and characterization.

We have purified and characterized topoisomerase I from Drosophila melanogaster. The molecular weight of the enzyme is 135,000; 100,000, 90,000, and 65,000 molecular weight products result from degradation of the enzyme. The enzyme relaxes both positive and negative supercoiled DNA. Mg++ is not absolutely required, but stimulates the enzymatic activity considerably.     

The purification and characterization of a glomerular-basement-membrane-degrading neutral proteinase from rat mesangial cells.

A neutral proteinase, capable of degrading gelatin, has been found in both an active and a latent form in the medium from the culture of rat mesangial cells. The latent form had an Mr of 80,000-100,000 and could be activated with either 4-aminophenylmercuric acetate or prolonged incubation at neutral pH. The active form of the enzyme was extensively purified. The estimated Mr of the purified enzyme on gel filtration was approximately 200,000, indicating that the active enzyme formed aggregates. However, analysis by SDS/polyacrylamide-gel electrophoresis under reducing conditions showed two protein bands, with Mr 68,000 and 66,000. Both proteins were found to contain proteolytic activity when run on SDS/substrate gels. The enzyme was inhibited by EDTA and 1,10-phenanthroline, but not by inhibitors for cysteine, serine or aspartic proteinases. The enzyme did not digest fibronectin, bovine serum albumin, proteoglycan or interstitial collagen. The enzyme degraded pepsin-solubilized placental type V collagen at 31 degrees C, whereas similarly solubilized type IV collagen was only degraded at higher temperatures. In addition, the neutral proteinase degraded native soluble type IV collagen. It also had activity on insoluble type IV collagen of glomerular basement membrane. The above properties suggest that the mesangial neutral proteinase belongs to the gelatinase group of metalloproteinases and that it may play a role in the normal turnover of extracellular glomerular matrix.     

Production,purification and characterization of an endopolygalacturonase from Mucor rouxii NRRL 1894

An extracellular polygalacturonase (PGase) from Mucor rouxii NRRL 1894 was purified to homogeneity by two chromatographic steps using CM-Sepharose and Superdex 75. The purified enzyme was a monomer with a molecular weight of 43100 Da and a pI of 6. The PGase was optimally active at 35 °C and at pH 4.5. It was stable up to 30 °C and stability of PGase decrease rapidly above 60 °C. The extent of hydrolysis of different pectins was decreased with increasing of degrees of esterification. Except Mn²⁺, all the examined metal cations showed inhibitory effects on the enzyme activity. The apparent K_m and V_max values for hydrolyze of polygalacturonic acid (PGA) were 1.88 mg/ml and 0.045 μmol/ml/min, respectively. The enzyme released a series of oligogalacturonates from polygalacturonic acid indicating that it had an endo-action. Its N-terminal sequence showed homologies with the endopolygalacturonase from the psychrophilic fungus Mucor flavus.     

Production, purification and characterization of an extracellular keratinase from Lysobacter NCIMB 9497

AIMS: The aim of this study was to determine the keratinolytic ability of a range of bacteria and subsequently, to characterize the keratinase showing the greatest biotechnological potential. METHODS AND RESULTS: Nine bacteria, reported to produce extracellular proteases, were screened for production of keratinases. Of these, Lysobacter NCIMB 9497 exhibited the highest keratinolytic activity. The keratinase from this strain (Mr 148 kDa) was purified and characterized. Optimum activity occurred at 50 degrees C; no inhibition was demonstrated by phenylmethylsulphonyl fluoride (PMSF), but inhibition by EDTA was demonstrated. CONCLUSIONS: This study indicates that keratinase is a metalloprotease with a high degree of keratinolytic activity and stability. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first detailed report of a metalloprotease with keratinolytic activity. The novel reaction mechanism, degree of keratinolytic activity and stability indicate considerable biotechnological potential in the leather industry, and in the processing of poultry waste.     

Production, rapid purification and catalytic characterization of extracellular phytase from Aspergillus ficuum

A rapid purification scheme utilizing three chromatographic steps resulted in 6 fold purification of Aspergillus ficuum phytase (myo-inositol-hexakisphosphate 3-phosphohydrolase, EC 3.1.3.8). At pH 5.0 and 60 degrees C the enzyme performed acceptably for 2.0 hr with only 30% diminished catalytic rate at the end. Substrate concentration exceeding 2mM was inhibitory. The inorganic orthophosphate, the product and a weak inhibitor, exhibited a Ki of 1.9 x 10(-3)M. The extracellular phytase has the potential for industrial use since it can be over produced, easily purified, remain catalytically active for a longer period and is not subjected to severe product inhibition.     

Production of human immunodeficiency virus from T-lymphoblastoid cells using serum-free medium

Summary A serum-free medium was constructed for the suspension culture of mammalian cells. Effects of the serum-free medium on the cultivation of T-lymphoblastoid cells secreting human immunodeficiency virus (HIV) were observed. The cell growth and HIV production in the serum-free medium were comparable to those in the medium supplemented with 10% fetal bovine serum (FBS), which showed a possibility of economical industrial application.     

Isolation and characterization of a metalloproteinase secreted by rat glioma cells in serum-free culture

The isolation of a metalloproteinase secreted by a rat glioma cell line (BT5C) in serum-free media is described. After affinity purification, the activity was present as a double band with Mr 86000 and 76000 both of which required CaCl2 for activity. The enzyme was able to degrade gelatin but not casein. It was unable to degrade native types I, III, IV and V collagens but their denatured counterparts were degraded. Using a radiolabel release assay the enzyme was inhibited by EDTA, 1:10 phenanthroline and TIMP confirming that it belongs to the family of metalloproteinases. Its activity was not affected by either serine or cysteine protease inhibitors. The proteinase was activated by APMA but was unaffected by trypsin treatment.     

Production, purification, and characterization of xylanase from a hyperxylanolytic mutant of Aspergillus ochraceus

Enhancement of the productivity of xylanase and beta-xy-losidase of Aspergillus ochraceus was investigated by multistep mutagenesis. The spores of the wild strain were subjected to UV and N-methyl-N-nitro-N-nitro-soguanidine (NTG). The hyperxylanolytic mutant (NG-13), which showed good clearing on the surface of the xylan-agar plate, secretes xylanase and beta-xylosidase at high levels during growth on commercial xylan and on agricultural wastes. Both liquid and solid state cultures were employed in the study for enzyme production. The xylanase from NG-13 was purified to homogeneity by ammonium sulfate precipitation and gel filtration. This purified enzyme showed a pH optimum of 6.0 and was stable in the range of pH 5 to 10. Prolonged stability of the enzyme was observed at 45 degrees C though its activity was maximal at 50 degrees C. The molecular weight of the enzyme was estimated to be 4.3 x 10(4) by SDS-polyacrylamide gel electrophoresis and 5 x 10(4) by gel filtration on Sephadex G-75. The kinetic data showed that the K(m) and V(max) values for xylan were 1 x 10(-3)M and 19.6 mumol/ min/mg protein, respectively. The enzyme was both more active and thermostable in the presence of K(+)and was inactivated by thiol reagents such as Hg(2+), p-hydroxymercuribenzoate (PHMB), 3', 5'-dithiobis (2'-nitrobenzoic acid) (DTNB), and N-ethylmaleimide (NEM).     

DF 31, a sperm decondensation factor from Drosophila melanogaster: purification and characterization.

We have purified to homogeneity a Drosophila protein which is able to decondense Xenopus sperm chromatin. This protein, which we have called DF 31, is a heat-stable phosphoprotein which displays a molecular weight of 31 kDa on SDS-PAGE, but which has an apparent molecular weight of > 200 kDa on gel filtration. We show that DF 31 decondenses sperm DNA by displacement of sperm-specific proteins. In addition to its sperm decondensation activity, DF 31 is also able to facilitate nucleosome loading on both decondensed sperm DNA and on naked DNA template. The reaction as catalysed by DF 31 is quite efficient; however, the nucleosomes appear to be loaded randomly onto the DNA, not in regular arrays. Although the mechanism by which DF 31 aids nucleosome loading is not yet clear, it most probably occurs through binding of DF 31 to core histones.     

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.