Purification of recombinant human growth hormone from CHO cell culture supernatant by Gradiflow preparative electrophoresis technology

Purification of recombinant human growth hormone (rhGH) from Chinese hamster ovary (CHO) cell culture supernatant by Gradiflow large-scale electrophoresis is described. Production of rhGH in CHO cells is an alternative to production in Escherichia coli, with the advantage that rhGH is secreted into protein-free production media, facilitating a more simple purification and avoiding resolubilization of inclusion bodies and protein refolding. As an alternative to conventional chromatography, rhGH was purified in a one-step procedure using Gradiflow technology. Clarified culture supernatant containing rhGH was passed through a Gradiflow BF200 and separations were performed over 60 min using three different buffers of varying pH. Using a 50 mM Tris/Hepes buffer at pH 7.5 together with a 50 kDa separation membrane, rhGH was purified to approximately 98% purity with a yield of 90%. This study demonstrates the ability of Gradiflow preparative electrophoresis technology to purify rhGH from mammalian cell culture supernatant in a one-step process with high purity and yield. As the Gradiflow is directly scalable, this study also illustrates the potential for the inclusion of the Gradiflow into bioprocesses for the production of clinical grade rhGH and other therapeutic proteins. (C) 2003 Elsevier Science (USA). All rights reserved.

Purification of the keratan sulfate proteoglycan expressed in prostatic secretory cells and its identification as lumican

BACKGROUND. Secretory epithelial cells of human prostate contain a keratan sulfate proteoglycan (KSPG) associated with the prostatic secretory granules (PSGs). The proteoglycan has not been identified, but like the PSGs, it is lost in the early stages of malignant transformation. METHODS. Anion exchange and affinity chromatography were used to purify KSPG from human prostate tissue. Enzymatic deglycosylation was used to remove keratan sulfate (KS). The core protein was isolated using 2D gel electrophoresis, digested in-gel with trypsin, and identified by peptide mass fingerprinting (PMF). RESULTS. The purified proteoglycan was detected as a broad smear on Western blots with an apparent molecular weight of 65-95 kDa. The KS moiety was susceptible to digestion with keratanase 11 and peptide N-glycosidase F defining it as highly sulfated and N-linked to the core protein. The core protein was identified, following deglycosylation and PMF, as lumican and subsequently confirmed by Western blotting using an anti-lumican antibody. CONCLUSIONS. The KSPG associated with PSGs in normal prostate epithelium is lumican. While the role of lumican in extracellular matrix is well established, its function in the prostate secretory process is not known. It's potential to facilitate packaging of polyamines in PSGs, to act as a tumor suppressor and to mark the early stages of malignant transformation warrant further investigation. (C) 2003 Wiley-Liss, Inc.

Physico-chemical characterization and synthesis of neuronally active alpha-conotoxins

The high speciFIcity of alpha-conotoxins for different neuronal nicotinic acetylcholine receptors makes them important probes for dissecting receptor subtype selectivity. New sequences continue to expand the diversity and utility of the pool of available alpha-conotoxins. Their identification and characterization depend on a suite of techniques with increasing emphasis on mass spectrometry and microscale chromatography, which have benefited from recent advances in resolution and capability. Rigorous physicochemical analysis together with synthetic peptide chemistry is a prerequisite for detailed conformational analysis and to provide sufficient quantities of alpha-conotoxins for activity assessment and structure-activity relationship studies.

Anogenital gland secretions of Lemur catta and Propithecus verreauxi coquereli: A preliminary chemical examination

Although prosimians are greatly olfaction-oriented, little is known about the specifics of how they use scent to communicate. In this preliminary study we attempted to delineate intra- and interspecific differences among the anogenital gland secretions of two lemur species (Lemur catta and Propithecus verreauxi coquereli) using gas chromatography-mass spectrometry (GC-MS). The results indicate that the two species are discernible through scent. Furthermore, we were able to identify reproductive status using this technique. The anogenital secretions of the different sexes in L. catta, though perhaps not P. v. coquereli, are chemically distinguishable. Given this information, it appears that at least some lemur species can use scent marks to determine species, sex, and reproductive status. (C) 2004 Wiley-Liss, Inc.

Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus Paecilomyces variotii

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 degrees C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 degrees C, with a t(50) of 45 min at 60 degrees C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl alpha-D-maltoside, methyl-alpha-D-glucopyranoside, pullulan, alpha- and beta-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in alpha-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-alpha-D-glucan glucohydrolase).

Purification and Biochemical Characterization of Thermostable Alkaline Phosphatases Produced by Rhizopus microsporus var. rhizopodiformis

The biochemical properties of the alkaline phosphatases (AIPs) produced by Rhizopus micro-sporus are described. High enzymic levels were produced within 1-2 d in agitated cultures with 1% wheat bran. Intra- and extracellular AlPs were purified 5.0 and 9.3x, respectively, by DEAE-cellulose and ConA-sepharose chromatography. Molar mass of 118 and 120 kDa was estimated by gel filtration for both forms of phosphatases. SDS-PAGE indicated dimeric structures of 57 kDa for both forms. Mn(2+), Na(+) and Mg(2+) Stimulated the activity, while Al(3+) and Zn(2+) activated only the extracellular form. Optimum temperature and pH for both phosphatases were 65 degrees C and pH 8.0, respectively. The enzymes were stable at 50 degrees C for at least 15 min. Hydrolysis of 4-nitrophenyl phosphate exhibited a K(m) 0.28 and 0.22 mmol/L, with upsilon(lim) 5.89 and 4.84 U/mg, for intra- and extracellular phosphatases, respectively. The properties of the reported AlPs may be suitable for biotechnological application.

Estimating the potential refolding yield of recombinant proteins expressed as inclusion bodies

Recombinant protein production in bacteria is efficient except that insoluble inclusion bodies form when some gene sequences are expressed. Such proteins must undergo renaturation, which is an inefficient process due to protein aggregation on dilution from concentrated denaturant. In this study, the protein-protein interactions of eight distinct inclusion-body proteins are quantified, in different solution conditions, by measurement of protein second virial coefficients (SVCs). Protein solubility is shown to decrease as the SVC is reduced (i.e., as protein interactions become more attractive). Plots of SVC versus denaturant concentration demonstrate two clear groupings of proteins: a more aggregative group and a group having higher SVC and better solubility. A correlation of the measured SVC with protein molecular weight and hydropathicity, that is able to predict which group each of the eight proteins falls into, is presented. The inclusion of additives known to inhibit aggregation during renaturation improves solubility and increases the SVC of both protein groups. Furthermore, an estimate of maximum refolding yield (or solubility) using high-performance liquid chromatography was obtained for each protein tested, under different environmental conditions, enabling a relationship between yield and SVC to be demonstrated. Combined, the results enable an approximate estimation of the maximum refolding yield that is attainable for each of the eight proteins examined, under a selected chemical environment. Although the correlations must be tested with a far larger set of protein sequences, this work represents a significant move beyond empirical approaches for optimizing renaturation conditions. The approach moves toward the ideal of predicting maximum refolding yield using simple bioinformatic metrics that can be estimated from the gene sequence. Such a capability could potentially screen, in silico, those sequences suitable for expression in bacteria from those that must be expressed in more complex hosts. (C) 2004 Wiley Periodicals, Inc.

Purification and characterization of a thermostable alpha-amylase produced by the fungus Paecilomyces variotii

An alpha-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The alpha-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pl value of 4.5. Temperature and pH optima were 60 degrees C and 4.0, respectively. The enzyme was stable for 1 h at 55 degrees C, showing a t(50) of 53 min at 60 degrees C. Starch protected the enzyme against thermal inactivation. The a-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K(m) of alpha-amylase on Reagen (R) and Sigma (R) starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to alpha-amylases from Bacillus sp. These results confirmed that the studied enzyme was an a-amylase ((1 -> 4)-alpha-glucan glucanohydrolase). (C) 2010 Elsevier Ltd. All rights reserved.

Purification and Biochemical Properties of a Glucose-Stimulated beta-D-Glucosidase Produced by Humicola grisea var. thermoidea Grown on Sugarcane Bagasse

The effect of several carbon sources on the production of mycelial-bound beta-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium, but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction medium stimulated beta-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single band in PAGE and SDS-PAGE. The beta-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57 and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50 degrees C, respectively. The purified enzyme was thermostable up to 60 min in water at 55 degrees C and showed half-lives of 7 and 14 min when incubated in the absence or presence of 50 mM glucose, respectively, at 60 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-galactopyranoside, p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-xylopyranoside, o-nitrophenyl-beta-D-galactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-beta-D-fucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose or xylose at concentrations from 25 to 200 mM. The addition of purified or crude beta-glucosidase to a reaction medium containing Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea beta-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.

Properties of a purified thermostable glucoamylase from Aspergillus niveus

A glucoamylase from Aspergillus niveus was produced by submerged fermentation in Khanna medium, initial pH 6.5 for 72 h, at 40A degrees C. The enzyme was purified by DEAE-Fractogel and Concanavalin A-Sepharose chromatography. The enzyme showed 11% carbohydrate content, an isoelectric point of 3.8 and a molecular mass of 77 and 76 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Bio-Sil-Sec-400 gel filtration, respectively. The pH optimum was 5.0-5.5, and the enzyme remained stable for at least 2 h in the pH range of 4.0-9.5. The temperature optimum was 65A degrees C and retained 100% activity after 240 min at 60A degrees C. The glucoamylase remained completely active in the presence of 10% methanol and acetone. After 120 min hydrolysis of starch, glucose was the unique product formed, confirming that the enzyme was a glucoamylase (1,4-alpha-d-glucan glucohydrolase). The K (m) was calculated as 0.32 mg ml(-1). Circular dichroism spectroscopy estimated a secondary structure content of 33% alpha-helix, 17% beta-sheet and 50% random structure, which is similar to that observed in the crystal structures of glucoamylases from other Aspergillus species. The tryptic peptide sequence analysis showed similarity with glucoamylases from A. niger, A. kawachi, A. ficcum, A. terreus, A. awamori and A. shirousami. We conclude that the reported properties, such as solvent, pH and temperature stabilities, make A. niveus glucoamylase a potentially attractive enzyme for biotechnological applications.

Purification and biochemical characterization of a novel alpha-glucosidase from Aspergillus niveus

An extracellular alpha-glucosidase produced by Aspergillus niveus was purified using DEAE-Fractogel ion-exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5% PAGE and 10% SDS-PAGE. The enzyme presented 29% of glycosylation, an isoelectric point of 6.8 and a molecular weight of 56 and 52 kDa as estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The enzyme showed typical alpha-glucosidase activity, hydrolyzing p-nitrophenyl alpha-d-glucopyranoside and presented an optimum temperature and pH of 65A degrees C and 6.0, respectively. In the absence of substrate the purified alpha-glucosidase was stable for 60 min at 60A degrees C, presenting t (50) of 90 min at 65A degrees C. Hydrolysis of polysaccharide substrates by alpha-glucosidase decreased in the order of glycogen, amylose, starch and amylopectin. Among malto-oligosaccharides the enzyme preferentially hydrolyzed malto-oligosaccharide (G10), maltopentaose, maltotetraose, maltotriose and maltose. Isomaltose, trehalose and beta-ciclodextrin were poor substrates, and sucrose and alpha-ciclodextrin were not hydrolyzed. After 2 h incubation, the products of starch hydrolysis measured by HPLC and thin layer chromatography showed only glucose. Mass spectrometry of tryptic peptides revealed peptide sequences similar to glucan 1,4-alpha-glucosidases from Aspergillus fumigatus, and Hypocrea jecorina. Analysis of the circular dichroism spectrum predicted an alpha-helical content of 31% and a beta-sheet content of 16%, which is in agreement with values derived from analysis of the crystal structure of the H. jecorina enzyme.

Heterologous expression of an Aspergillus niveus xylanase GH11 in Aspergillus nidulans and its characterization and application

A xylanase was cloned from Aspergillus niveus and successfully expressed in Aspergillus nidulans (XAN). The full-length gene consisted of 890 bp and encoded 275 mature amino acids with a calculated mass of 31.3 kDa. The deduced amino acid sequence was highly homologous with the xylanase belonging to family 11 of the glycoside hydrolases. The recombinant protein was purified to electrophoretic homogeneity by anion-exchange chromatography and gel filtration. The optima of pH and temperature for the recombinant enzyme were 5.0 and 65 degrees C, respectively. The thermal stability of the recombinant xylanase was extremely improved by covalent immobilization on glyoxyl agarose with 91.4% of residual activity after 180 min at 60 degrees C, on the other hand, the free xylanase showed a half-life of 9.9 min at the same temperature. Affinity chromatography on Concanavalin A- and Jacalin-agarose columns followed by SDS-PAGE analyses showed that the XAN has O- and N-glycans. XAN promotes hydrolysis of xylan resulting in xylobiose, xylotriose and xylotetraose. Intermediate degradation of xylan resulting in xylo-oligomers is appealing for functional foods as the beneficial effect of oligosaccharides on gastrointestinal micro flora includes preventing proliferation of pathogenic intestinal bacteria and facilitates digestion and absorption of nutrients. (C) 2011 Elsevier Ltd. All rights reserved.

Microcystin production by a freshwater spring cyanobacterium of the genus Fischerella

We investigated the production of a hepatotoxic, cyclic heptapeptide, microcystin, by a filamentous branched cyanobacterium belonging to the order Stigonematales, genus Fischerella. The freshwater Fischerella sp. strain CENA161 was isolated from spring water in a small concrete dam in Piracicaba, Sao Paulo State, Brazil, and identified by combining a morphological description with 16S rRNA gene sequencing and phylogenetic analysis. Microcystin (MCYST) analysis performed using an ELISA assay on cultured cells gave positive results. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis detected 33.6 mu g MCYST-LR per gram dry weight of cyanobacterial cells. Microcystin profile revealed by quadrupole time-of-flight tandem mass spectrometry (Q-TOF-MS/MS) analysis confirmed the production of MCYST-LR. Furthermore, genomic DNA was analyzed by PCR for sequences similar to the ketosynthase (KS) domain of the type I polyketide synthase gene, which is involved in microcystin biosynthesis. This revealed the presence of a KS nucleotide fragment similar to the mcyD and ndaD genes of the microcystin and nodularin synthetase complexes. Phylogenetic analysis grouped the Fischerella KS sequence together with mcyD sequences of the three known microcystin synthetase operon (Microcystis, Planktothrix and Anabaena) and ndaD of the nodularin synthetase operon, with 100% bootstrap support. Our findings demonstrate that Fischerella sp. CENA161 produces MYCST-LR and for the first time identify a nucleotide sequence putatively involved in microcystin synthesis in this genus. (C) 2009 Elsevier Ltd. All rights reserved.

Bioprocess-centered molecular design (BMD) for the efficient production of an interfacially active peptide

The efficient expression and purification of an interfacially active peptide (mLac21) was achieved by using bioprocess-centered molecular design (BMD), wherein key bioprocess considerations are addressed during the initial molecular biology work. The 21 amino acid mLac21 peptide sequence is derived from the lac repressor protein and is shown to have high affinity for the oil-water interface, causing a substantial reduction in interfacial tension following adsorption. The DNA coding for the peptide sequence was cloned into a modified pET-31(b) vector to permit the expression of mLac21 as a fusion to ketosteroid isomerase (KSI). Rational iterative molecular design, taking into account the need for a scaleable bioprocess flowsheet, led to a simple and efficient bioprocess yielding mLac21 at 86% purity following ion exchange chromatography (and >98% following chromatographic polishing). This case study demonstrates that it is possible to produce acceptably pure peptide for potential commodity applications using common scaleable bioprocess unit operations. Moreover, it is shown that BMD is a powerful strategy that can be deployed to reduce bioseparation complexity. (C) 2004 Wiley Periodicals, Inc.

Activity of platinum-tin catalysts prepared by the Pechini-Adams method for the electrooxidation of ethanol

Pt-Sn electrocatalysts of different compositions were prepared and dispersed on carbon Vulcan XC-72 using the Pechini-Adams method. The catalysts were characterized by energy dispersive X-ray analysis and X-ray diffraction. The electrochemical properties of these electrode materials were also examined by cyclic voltammetry and chronoamperometric experiments in acid medium. The results showed that the presence of Sn greatly enhances the activity of Pt towards the electrooxidation of ethanol. Moreover, it contributes to reduce the amount of noble metal in the anode of direct alcohol fuel cells, which remains one of the challenges to make the technology of direct alcohol fuel cells possible. Electrolysis of ethanol solutions at 0.55 V vs. RHE allowed to determine by liquid chromatography acetaldehyde and acetic acid as the main reaction products. CO(2) was also analyzed after trapping it in a NaOH solution indicating that the cleavage of the C-C bond in the ethanol molecule did occur during the adsorption process. In situ IR reflectance spectroscopy helped to investigate in more details the reaction mechanism through the identification of the reaction products as well as the presence of some intermediate adsorbed species, such as linearly bonded carbon monoxide. (C) 2009 Elsevier B.V. All rights reserved.