Purification of bovine pancreatic glucagon as a by-product of insulin production

A process for purifying bovine pancreatic glucagon as a by-product of insulin production is described. The glucagon-containing supernatant from the alkaline crystallization of insulin was precipitated using ammonium sulfate and isoelectric precipitation. The isoelectric precipitate containing glucagon was then purified by ion-exchange chromatography on Q-Sepharose FF, gel filtration on Sephadex G-25 and ion-exchange chromatography on S-Sepharose FF. A pilot scale test was performed with a recovery of 87.6% and a purification factor of 8.78 for the first chromatographic step, a recovery of 75.1% and a purification factor of 3.90 for the second, and a recovery of 76.2% and a purification factor of 2.36 for the last one. The overall yield was 50%, a purification factor of 80.8 was obtained and the fraction containing active glucagon (suitable for pharmaceutical preparations) was 84% pure as analyzed by HPLC.     

Overexpression and large-scale purification of recombinant hamster polymorphic arylamine N-acetyltransferase as a dihydrofolate reductase fusion protein

N-Acetyltransferases (NATs) are enzymes that catalyze the detoxification and/or bioactivation of a variety of xenobiotics. Rapid kinetic, biophysical, structural, and bioactivation studies on NATs require quantities of purified enzyme capable of being obtained only through recombinant DNA technology. This laboratory has previously developed a protein expression and purification system in which NATs are expressed as proteins fused to a FLAG octapeptide followed by a thrombin-cleavage site to allow liberation of the rNAT. Typically, however, only 0.5-1.5 mg of the recombinant NAT's could be readily purified in a single isolation sequence by immunoaffinity chromatography. Therefore, the expression system was modified by inserting the L54F dihydrofolate reductase (DHFR) mutant gene sequence between the FLAG octapeptide and the thrombin-cleavage site. Expression was carried out with TOPP3 Escherichia coli cells. The new purification methodology utilizes the unique pH dependence of binding to a methotrexate (MTX)-affinity column by the L54F DHFR mutant. Unfortunately, the affinity chromatography strategy did not work satisfactorily. Although the specific activity of the purified rNAT2 was comparable to that of NAT2 obtained from hamster tissue, only 3% of the activity was recovered. The apparent cause of the low recovery is the unanticipated irreversible binding of rNAT2 to MTX. Ion-exchange chromatography was investigated as an alternative purification method. An initial DEAE anion-exchange column resulted in partial purification of the fusion protein. The fusion protein was cleaved with thrombin and reapplied to a DEAE anion-exchange column. The second DEAE column resulted in not only the separation of rNAT2-70D from FLAG-L54F DHFR, but also the purification of rNAT2-70D to near homogeneity. Application of the nearly homogeneous rNAT2-70D to a gel-filtration column resulted in recovery of homogeneous protein. The ion-exchange method of purifying rNAT2-70D is inexpensive and simple and yields more than 8 mg of pure enzyme from 1 liter of cell culture.     

Reactivity of haloketenes and halothioketenes with nucleobases: reactions in vitro with DNA

A novel secondary metabolite SCH 42282 (1), with antifungal activity was isolated from the fermentation broth of a soil actinomycete identified as a Microtetraspora sp. The active compound was separated from the fermentation broth by butanol extraction and purified on a silica gel column and by multi-coil counter current chromatography. The compound was identified as a novel macrolactam trisaccharide related to SCH 38518 (4). The structure was established by hydrolysis of the parent compound and spectroscopic studies of the acetate derivative. The compound is active against Candida spp. (Geometric Mean MIC's. 18 micrograms/ml) but less active SCH 42729 (3). the disaccharide (Geometric Mean MIC's, > or = 10.7 micrograms/ml and SCH 38518 (4), the monosaccharide (Geometric Mean Mic's, 3.8 micrograms/ml.     

Three-dimensional fibroblast culture implant for the treatment of diabetic foot ulcers: metabolic activity and therapeutic range

Dialysis techniques are discussed as a means for effective removal of low-molecular-mass components from fermentation broth to reach high cell density. Reactor systems and process strategies, the relevant properties of membranes and examples for high-density fermentation with dialysis, and problems related to scale-up are addressed. The dialysis technique has turned out to be very efficient and reliable for obtaining high cell densities. As in dialysis processes the membranes are not perfused, membrane clogging is not a problem as it is for micro- and ultrafiltration. By applying a "nutrient-split" feeding strategy, the loss of nutrients can be avoided and the medium is used very efficiently. The potential of dialysis cultures is demonstrated on the laboratory scale in a membrane dialysis reactor with an integrated membrane and in reactor systems with an external dialysis loop. In dialysis cultures with different microorganisms (Staphylococci, Escherichia coli, extremophilic microorganisms, Lactobacilli) the cell densities achieved were up to 30 times higher than those of other fermentation methods. The technique enables high cell densities to be attained without time-consuming medium optimization. For animal cell cultures the concept of a fixed bed coupled with dialysis proved to be very effective.     

Pichia stipitis genes for alcohol dehydrogenase with fermentative and respiratory functions

Two genes coding for isozymes of alcohol dehydrogenase (ADH); designated PsADH1 and PsADH2, have been identified and isolated from Pichia stipitis CBS 6054 genomic DNA by Southern hybridization to Saccharomyces cerevisiae ADH genes, and their physiological roles have been characterized through disruption. The amino acid sequences of the PsADH1 and PsADH2 isozymes are 80.5% identical to one another and are 71.9 and 74.7% identical to the S. cerevisiae ADH1 protein. They also show a high level identity with the group I ADH proteins from Kluyveromyces lactis. The PsADH isozymes are presumably localized in the cytoplasm, as they do not possess the amino-terminal extension of mitochondrion-targeted ADHs. Gene disruption studies suggest that PsADH1 plays a major role in xylose fermentation because PsADH1 disruption results in a lower growth rate and profoundly greater accumulation of xylitol. Disruption of PsADH2 does not significantly affect ethanol production or aerobic growth on ethanol as long as PsADH1 is present. The PsADH1 and PsADH2 isozymes appear to be equivalent in the ability to convert ethanol to acetaldehyde, and either is sufficient to allow cell growth on ethanol. However, disruption of both genes blocks growth on ethanol. P. stipitis strains disrupted in either PsADH1 or PsADH2 still accumulate ethanol, although in different amounts, when grown on xylose under oxygen-limited conditions. The PsADH double disruptant, which is unable to grow on ethanol, still produces ethanol from xylose at about 13% of the rate seen in the parental strain. Thus, deletion of both PsADH1 and PsADH2 blocks ethanol respiration but not production, implying a separate path for fermentation.     

Monoclonal antibody production with on-line harvesting and process monitoring

A semi-automated system has been designed for on-line harvesting and monitoring of monoclonal antibody (mAb) production. [The antibody was directed against the peptide AGPAGTGKTTKDL.] Analytical and purification units were interfaced to the fermenter via a hollow fiber cartridge in which fermentation broth was continuously circulated through the lumen of the hollow fiber system. Permeate from the hollow fiber cartridge was pumped through either an analytical sampling loop or a preparative Protein G column where antibody species were captured. Switching between monitoring and harvesting was achieved by two 3-way toggle valves. Samples from the analytical sampling loop were transported to an analytical Protein G chromatography column for quantitation of all immunoglobulin G species in the fermenter. Data acquisition and processing was performed by the data system of the liquid chromatograph. All valves in the system except the two toggle valves were controlled by the liquid chromatograph. Antibody biosynthesis was monitored for the first 60 h of fermentation. Harvesting was initiated when mAb accumulated in the fermenter. Complete harvesting took approximately 90 h.     

Biochemistry of Vibrio cholerae virulence: purification of cholera enterotoxin by preparative disc electrophoresis

Procedures for cholera enterotoxin purification previously developed in this labarotory were not applicable to large-scale purification, and these methods resulted in low yields of pure toxin. An efficient scheme has been developed whereby pure cholera enterotoxin can be obtained from 6 to 8 liters of culture supernatant fluid. This method consists of concentration by membrane ultrafiltration followed by gel filtration and cation-exchange chromatography. Pure cholera enterotoxin of high biological potency was obtained after a final step of preparative acrylamide gel electrophoresis. The degree of purity of the toxin-antigen as well as its biological activity were determined at various setps of purification. This alternate technique for purification is offered because of the widespread interest in cholera enterotoxin as a specific stimulator of adenyl cyclase.     

Studies on the safety of glucose and paraben-containing neostigmine for intrathecal administration

Oxysterols are biologically active molecules generated during oxidation of LDL. Several of these oxysterols were found in macrophage-derived foam cells from human atherosclerotic tissue (eg, 7-hydroxycholesterol, 7-ketocholesterol, 5-epoxycholesterol, and 25-hydroxycholesterol). A specific stimulation of interleukin-8 (IL-8) production by oxidized LDL (oxLDL) has been shown by other investigators. In foam cells from human atherosclerotic tissue, we found high levels of IL-8 (183.1 pg/10(6) cells) compared with monocytes (23.2 pg/10(6) cells) or monocyte-derived macrophages in culture (1.5 pg/10(6) cells). When monocytes and monocyte-derived macrophages, in vitro, were exposed to a series of different oxysterols, we found that all oxysterols tested had a tendency to stimulate IL-8 production but that 25-hydroxycholesterol was the most potent one. This stimulation of IL-8 production was time and dose dependent and could be blocked by cycloheximide. These results indicate that oxysterols in oxLDL may have a regulatory effect on IL-8 production. IL-8, a potent chemoattractant, may play a role in the recruitment of T lymphocytes and smooth muscle cells into the subendothelial space and may contribute to the formation of atherosclerotic lesions.     

Fermentation studies of rustmicin production by a Micromonospora sp

The antifungal antibiotic rustmicin was detected in the fermentation broth of the actinomycete MA 7094 as a specific inhibitor of sphingolipid biosynthesis in Candida albicans and as a potent fungicidal agent against Cryptococcus neoformans. Taxonomic characterization by both classical means and PCR fingerprinting supported the assignment of the producing culture to the genus Micromonospora. Fermentation medium optimization studies showed that the concentration of tomato paste in the medium was critical to increased production of rustmicin by MA 7094. The stimulatory effect of tomato paste in the medium on rustmicin production appeared to be related to the maintenance of pH at or below a value of 6.0. Addition of the antifoam agent P-2000 to the fermentation was found to dramatically reduce the rustmicin titer, while substitution of another antifoam agent, UCON-LB625, resulted in a 100% increase in the amount of rustmicin detected. After fermentation optimization studies and the generation of a non-sporulating mutant of MA 7094, the rustmicin titer was increased from an initial titer of 10mg/liter to 145 mg/liter.     

Bacillus Thuringiensis Fermentation of Primary and Mixed Sludge: Rheology and Process Performance

Bench scale fermentation of primary, mixed, and secondary (raw and hydrolyzed) sludge and starch industry wastewater was carried out using Bacillus thuringiensis (Bt) var. kurstaki HD-1 to test their feasibility as potential growth substrates on the basis of rheology and process performance in comparison to soyameal (semisynthetic) commercial medium. All fermented media exhibited pseudoplastic pattern, followed power law for viscosity with greater shear thinning for primary sludge (raw and hydrolyzed). Improved rheology correlated well with the fermented broth morphology. The total cell and viable spore counts, oxygen consumption, maximum specific growth rate (μmax), and entomotoxicity were, respectively, 2, 4, 1.5–2, and 3 folds lower in primary sludge in contrast to mixed sludge, rendering primary sludge unsuitable as raw material for Bt fermentation and eventual formulation. Further, the rheology studies of secondary sludge and starch industry wastewater proved them to be good Bt fermentation alternatives.     

Aggregation of recombinant hepatitis B surface antigen in Pichia pastoris

The combination of immunoaffinity and size-exclusion chromatography (SEC) is a powerful tool to analyze multiprotein particle assembly. This approach was used to investigate the source of aggregation of recombinant hepatitis B surface antigen (HBsAg) detected in purified material. As HBsAg aggregation does not originate in the stresses, such as the concentration of HBsAg solutions, temperature and chaotropic agents, it is less probable that the HBsAg aggregate is produced during the process. To test whether aggregation takes place in vivo, crude yeast extract containing the expressed HBsAg was fractioned on a Sephacryl S-400 column just after cell disruption, and each fraction immunopurified individually. As a result, the HBsAg aggregate was isolated from a fraction corresponding to the elution of large particle aggregates only, not native HBsAg particles. It was biologically active, which demonstrates aggregate formation by specific assembly of partially or wholly folded HBsAg intermediates.     

High-level expression of biologically active, soluble forms of ICAM-1 in a novel mammalian-cell expression system

LFA-1/ICAM-1 interaction is important in facilitating a number of cellular events including antigen-specific T-cell activation and leukocyte transendothelial migration. We are interested in defining residues and contact sites that mediate ICAM-1 interaction with the integrin receptor, LFA-1. To provide sufficient material to facilitate study of the interaction of this ligand-receptor pair, we have developed a new high-level mammalian-cell expression system based on the use of the herpes simplex virus (HSV) VP16 transactivator and the HSV IE175 promoter to direct expression of foreign genes in BHK cells. In this system, the gene of interest is expressed as a fusion protein with a carboxyl terminal decapeptide tail to aid in identification, quantitation, and affinity purification of recombinant protein. This system allowed rapid generation of cell lines producing high levels of levels of soluble proteins corresponding to the full-length extracellular (sICAM453) and the amino terminal two immunoglobulin domains (sICAM185) of ICAM-1. Both sICAM453 and sICAM185 were biologically active and were purified in a single step from conditioned media by antibody affinity chromatography.     

Mutational analysis of a satellite phage activator

A culture medium, named olive juice broth, which resembles the natural environment of Lactobacillus plantarum in the traditional Spanish-style green olive fermentation was obtained from green olives. In this medium, the bacteriocin-producing L. plantarum LPCO10 strain was able to produce bacteriocin throughout the incubation time (15 days). Bacteriocin purification from olive juice broth was achieved by a protocol including ammonium sulphate precipitation of cell-free, L. plantarum LPCO10 culture supernatants, and cation-exchange, hydrophobic-interaction and reversed-phase chromatographies. In a series of mixed cultures in olive juice broth, L. plantarum LPCO10 was able to dominate the bacteriocin-sensitive L. plantarum 128/2 strain, whereas the non-bacteriocin-producing, LPCO10 strain derivative, L. plantarum 55-1 strain did not show such capability. These results indicated that olive juice broth may be a valuable experimental substitute for olive fermentation brine in gaining more knowledge about the role of the bacteriocin-producing L. plantarum strains in the control of the Spanish-style green olive fermentation.     

Interleukin 1 (IL-1) and IL-1-receptor antagonist (IL-1-RA) in middle ear cholesteatoma: an analysis of protein production and biological activity

Cytokine networks are now presumed to play an essential role in the pathogenesis of middle ear cholesteatoma. Of the factors identified in cholesteatoma, interleukin-I (IL-1)-alpha appears to be especially important because of its stimulation of keratinocyte proliferation as well induction of bone resorption. To further characterize the possible role of IL-1 in the pathogenesis of cholesteatoma, we quantified the levels of IL-1 and IL-1-receptor antagonist (IL-1-RA) present using the bicinchonic acid protein assay and enzyme-linked immunosorbent assay (ELISA) on tissue extracts from 20 cholesteatoma specimens. The presence of biologically active IL-1 was also analyzed, using the cell line LBRM-33 and an ELISA for the detection of interleukin-2 (IL-2). Human skin obtained from the external ear canal was used as control. The amounts of IL-1-alpha in cholesteatoma (34.9 +/- 19.5) were higher than in human skin (6.7 +/- 2.8). The observed differences were statistically significant by Student's t-test (P < 0.01). Skin samples showed elevated concentrations of IL-1-RA (248.3 +/- 30.2) in comparison to that in the cholesteatoma (80.8 +/- 13.5). This was also statistically significant (P < 0.01). Whereas IL-1 activity was not detected in skin samples, all cholesteatoma specimens studied showed a stimulation effect on the production of IL-2 when incubated with the cell line LBRM-33. The results point to an over-expression of IL-1 concurrent with a decreased secretion of IL-1-RA in middle ear cholesteatoma. Furthermore IL-1-RA production is deficient relative to total IL-1 production, resulting in the presence of active IL-1.     

Arrested rearrangement of TCR V beta genes in thymocytes from children with X-linked severe combined immunodeficiency disease

Human X-linked severe combined immunodeficiency disease (SCID) is an immunodeficiency disorder in which T cell development is arrested in the thymic cortex. B lymphocytes in children with X-linked SCID seem to differentiate normally. X-linked SCID is associated with a mutation in the gene that encodes the IL-2R gamma-chain. Because TCR-beta gene recombination is a pivotal initial event in T lymphocyte ontogeny within the thymus, we hypothesized that a failure to express normal IL-2R gamma could lead to impaired TCR-beta gene recombination in early thymic development. PCR was used to determine the status of TCR-beta gene-segment rearrangements in thymic DNA that had been obtained from children with X-linked SCID. The initial step in TCR-beta gene rearrangement, that of D beta to J beta recombination, was readily detected in all thymus samples from children with X-linked SCID; in contrast, V beta to DJ beta gene rearrangements were undetectable in the same samples. Both D beta to J beta and V beta to DJ beta TCR genes were rearranged in the thymic tissues obtained from immunologically normal children. We conclude that TCR beta-chain gene rearrangement is arrested in children with X-linked SCID. Our results suggest a causative relationship between the failure of TCR beta-chain gene rearrangements to proceed beyond DJ beta rearrangements and the production of a nonfunctional IL-2R gamma-chain.     

Insulin-degrading enzyme regulates extracellular levels of amyloid beta-protein by degradation

Excessive cerebral accumulation of the 42-residue amyloid beta-protein (Abeta) is an early and invariant step in the pathogenesis of Alzheimer's disease. Many studies have examined the cellular production of Abeta from its membrane-bound precursor, including the role of the presenilin proteins therein, but almost nothing is known about how Abeta is degraded and cleared following its secretion. We previously screened neuronal and nonneuronal cell lines for the production of proteases capable of degrading naturally secreted Abeta under biologically relevant conditions and concentrations. The major such protease identified was a metalloprotease released particularly by a microglial cell line, BV-2. We have now purified and characterized the protease and find that it is indistinguishable from insulin-degrading enzyme (IDE), a thiol metalloendopeptidase that degrades small peptides such as insulin, glucagon, and atrial natriuretic peptide. Degradation of both endogenous and synthetic Abeta at picomolar to nanomolar concentrations was completely inhibited by the competitive IDE substrate, insulin, and by two other IDE inhibitors. Immunodepletion of conditioned medium with an IDE antibody removed its Abeta-degrading activity. IDE was present in BV-2 cytosol, as expected, but was also released into the medium by intact, healthy cells. To confirm the extracellular occurrence of IDE in vivo, we identified intact IDE in human cerebrospinal fluid of both normal and Alzheimer subjects. In addition to its ability to degrade Abeta, IDE activity was unexpectedly found be associated with a time-dependent oligomerization of synthetic Abeta at physiological levels in the conditioned media of cultured cells; this process, which may be initiated by IDE-generated proteolytic fragments of Abeta, was prevented by three different IDE inhibitors. We conclude that a principal protease capable of down-regulating the levels of secreted Abeta extracellularly is IDE.