bluuebunny/crossref_metadata_2025 huggingface dataset:
Alkaline protease from Spilosoma obliqua: potential applications in bio‐formulations
Authors: Anwar Adil , Saleemuddin Mohammed
Published: November 2003

Some properties of the purified alkaline protease from larvae of the insect Spilosoma obliqua (Lepidoptera) and its potential application as an additive in various bio‐formulations are reported. The novel feature of the present study is the use of insect protease. The protease was found to be compatible with some of the commercial detergents tested, and was also effective in cleaving various protein substrates tested, albeit to different extents, implying broader substrate specificity and effectiveness of the protease against a wide variety of stains. This property of the protease can also be exploited by using it as an active component in enzymic debriders in view of its ability to digest various protein substrates. The insect protease appears to be potentially useful as an additive in detergent, stain remover and other bio‐formulations.
Efficient utilization of starch by a recombinant strain of Saccharomyces cerevisiae producing glucoamylase and isoamylase
Authors: Ma Yih‐Jer , Lin Long‐Liu , Chien Hungchien Roger , Hsu Wen‐Hwei
Published: November 2003

Two plasmids, designated pRTI and pTI, were constructed to allow the integration of a bacterial isoamylase gene (iso) into Saccharomyces cerevisiae G23‐8 chromosome. The integrative plasmid pRTI comprises the iso gene from Pseudomonas amyloderamosa, a portion of S. cerevisiae ribosomal DNA (rDNA), S. cerevisiae trp1 gene deficient in promoter and the bacterial vector pSP72. The structure of plasmid pTI is similar to that of pRTI, except that it lacks an rDNA segment. The Aspergillus awamori glucoamylase and P. amyloderamosa isoamylase genes were expressed in the recombinant strain of S. cerevisiae under the control of the yeast alcohol dehydrogenase gene (adh1) promoter. Southern‐blot analysis showed that these plasmids were integrated into the yeast chromosome in tandem repeat and dispersion copies. The recombinant strains could assimilate starch more efficiently than the recipient strain with a conversion rate of greater than 95%.
Characterization of N‐linked oligosaccharides bearing sialyl Lewis x moieties on an alternatively glycosylated form of soluble complement receptor type 1 (sCR1)
Authors: Picard Michele D. , Pettey Carolyn L. , Marsh Henry C. , Thomas Lawrence J.
Published: November 2003

We sought to produce a complement inhibitory protein possessing oligosaccharides specifically modified to contain the sialyl Lewis x (sLex) moiety. This modified glycoprotein could combine anti‐complement activity with the ability to inhibit selectin‐mediated interactions and concentrate this activity to sites of activated endothelium where selectins are upregulated. Soluble complement receptor type 1 (sCR1), previously shown to be effective in inhibiting the complement cascade, was produced in a cell line capable of adding fucose to N‐linked oligosaccharides in the α1‐3 linkage, which is necessary for sLex glycosylation. The glycoprotein purified from these cells was designated sCR1sLex, and may prove to be more effective than sCR1 in some clinical applications. Detailed analysis and characterization of sCR1sLex was performed to confirm that the N‐linked oligosaccharides possessed sLex moieties and also to determine the extent of sLex glycosylation. The glycoproteins were characterized by oligosaccharide profiling, sequencing, linkage analysis and quantified by differential enzymic digestion, using fluorophore‐assisted carbohydrate electrophoresis. The major glycans were identified as biantennary oligosaccharides (including sialylated and non‐core fucosylated glycans). The linkages of sialic acid and the branched fucose were analysed by digestion with linkage‐specific enzymes and subsequent separation by electrophoresis. All data were consistent with the presence of sLex moieties on the N‐linked oligosaccharides of sCR1sLex. sCR1sLex is a prime example of a recombinant protein expressed with oligosaccharides engineered for a specific biological function, and produced using a commercially viable method.
High‐level expression of tetanus toxin fragment C–thioredoxin fusion protein in Escherichia coli
Authors: Ribas Adriana V. , Ho Paulo L. , Tanizaki Martha M. , Raw Isaias , Nascimento Ana L. T. O.
Published: November 2003

An insert of Clostridium tetani DNA corresponding to fragment C of tetanus toxin was amplified by PCR. This 1.4 kb fragment was cloned into the high‐expression vector pET32a, under control of the T7 promoter. Expression of this plasmid in Escherichia coli BL21(DE3) resulted in the production of a fusion protein (≈62 kDa) consisting of 112 amino acids of thioredoxin and ≈450 amino acids of fragment C. This fusion protein was recognized by anti‐tetanus toxoid antiserum in an ELISA and on immunoblots. The recombinant fragment‐C‐thioredoxin protein was purified significantly in one step by Ni2+‐chelate Sepharose, the final yield being ≈35 mg/l. Immunization of animals with the recombinant protein produced antibodies that were able to recognize the tetanus toxin. By using this gene‐fusion expression system we produced soluble fragment C of tetanus toxin in a high yield, preventing many problems inherent in the use of other expression systems that produce either insoluble fragment C in inclusion bodies, or a soluble form, but in low yield, using E. coli as the expression host.
Relationship between conformational stability and lyophilization‐induced structural changes in chymotrypsin
Authors: Carrasquillo Karen G. , Sanchez Cheryll , Griebenow Kai
Published: November 2003

The relationship between protein conformational stability in aqueous solution and the magnitude of lyophilization‐induced structural changes was investigated employing α‐ and γ‐chymotrypsin. As a measure of the conformational stability the melting temperature Tm was determined in distilled water at various pH values. The proteins were then lyophilized from those pH values where the conformational stability was maximum (pH 4.5) and minimum (pH 7.8). Protein secondary structure was quantitatively determined utilizing Fourier‐transform infrared spectroscopy employing two regions sensitive to protein structure, the amide‐I (1600–1700 cm−1) and amide‐III (1215–1335 cm−1). Lyophilization induced significant structural alterations in both proteins, characterized by a slight decrease in the α‐helix and a significant increase in the β‐sheet content. However, regardless of the pH from which the proteins were lyophilized, the secondary structures in the solid state were indistinguishable. This result shows that there is no relationship between the conformational stability in aqueous solution and the magnitude of lyophilization‐induced structural changes. We also investigated whether lyoprotectants could minimize lyophilization‐induced structural changes by increasing protein conformational stability in aqueous solution. After having identified trehalose as being efficient in largely preventing lyophilization‐induced structural alterations, we conducted co‐lyophilization experiments from various pH values. The results obtained exclude any contribution from increased protein conformational stability caused by the additive in aqueous solution to the beneficial structural preservation upon lyophilization. This can be understood because the dehydration and not the freezing process, as shown in an air‐drying experiment, mainly causes protein structural alterations.
Immobilization of Pseudomonas cepacia lipase in a phyllosilicate sol–gel matrix: effectiveness as a biocatalyst
Authors: Hsu An‐Fei , Foglia Thomas A. , Shen Siyan
Published: November 2003

A novel procedure is described for immobilizing a lipase from Pseudomonas cepacia (PS‐30) within a phyllosilicate sol–gel matrix. The method is based on cross‐linking a phyllosilicate clay with silicate polymers produced by the controlled hydrolysis of tetramethyl orthosilicate (TMOS). The activity of the phyllosilicate sol–gel‐immobilized lipase was dependent upon the type of alkylammonium salt, inorganic catalyst and volume ratio of phyllosilicate clay to TMOS used. Lipase PS‐30 immobilized in this way was more stable and had higher activity compared with the free lipase. Studies on the lipase‐catalysed esterification of lauric acid with octan‐1‐ol in iso‐octane showed that under controlled water activity conditions the phyllosilicate sol–gel‐immobilized lipase had better activity compared with other supported lipase preparations. In addition, the phyllosilicate sol–gel‐immobilized lipase was reusable for at least five esterification cycles without significant loss of activity.
Polyclonal‐antibody‐mediated insolubilization and stabilization of papain
Authors: Khan Shakil A. , Iqbal Jawaid
Published: November 2003

Antisera raised in rabbits against native iodoacetamide‐ and iodoacetic acid‐modified papain contained precipitating and non‐inhibitory antibodies. Papain could be insolubilized as enzyme–antibody adducts with the γ‐globulin fraction derived from the antiserum. Two insolubilized papain preparations, designated A and B, with low and high antibody/enzyme ratios, respectively, exhibited high enzyme activity and markedly enhanced thermal and denaturant stabilities. However, papain antibody adduct B was superior in activity and stability over preparation A. The usefulness of immobilized papain in biopharmaceutical and other industries is also discussed.
Characterization of Mucor pusillus rennin expressed in Pichia pastoris: enzymic, spectroscopic and calorimetric studies
Authors: Beldarraín Alejandro , Acosta Niuris , Montesinos Raquel , Mata Marlene , Cremata José
Published: November 2003

The aspartic proteinase gene of Mucor pusillus rennin expressed in Pichia pastoris was characterized in terms of structural and conformational stability induced by temperature. This enzyme is 12% glycosylated, with a similar specific activity to the native fungal enzyme. The secondary structure determined by CD is mainly due to β‐sheet structures with an important contribution of aromatic components. The calorimetric studies were carried out in the temperature range in which the enzyme is most stable. The enzyme undergoes an irreversible, highly scan‐rate‐dependent thermal denaturation under all the experimental conditions investigated. Between pH 3.0 and 7.0, only one endotherm characterized the thermal denaturation of enzyme. At pH 5.0, the most stable condition found, the denaturation can be fitted to the two‐state irreversible model. Thus the kinetic constant and activation parameters of the denaturation process could be obtained. Upon reaching pH 7.5, the denaturation is characterized by two endotherms. This evidence indicates the complex tridimentional structure of this enzyme. Finally, taking into account the conservative tertiary structure of the aspartic proteinase family we comment on our results with reference to the crystallographic structure of M. pusillus pepsin [Newman, Watson, Roychowdhury, Badasso, Cleasby, Wood, Tickle and Blundell (1993).
Adsorption of plasma proteins on to poly(ethylene oxide)/poly(propylene oxide) triblock copolymer films: a focus on fibrinogen
Authors: O’Connor Stephen M. , DeAnglis Ashley P. , Gehrke Stevin H. , Retzinger Gregory S.
Published: November 2003

Triblock copolymers of the form PEOαPPOβPEOα [where PEO is poly(ethylene oxide) and PPO is poly(propylene oxide)] have many biomedical applications, many of which depend on the surface properties of the copolymers and the influence that those properties have on the adsorption of proteins. As a tool to help us better understand, predict and exploit the influence of these triblock copolymers on protein adsorption, we developed a model system in which well‐defined monolayers of the copolymers are supported by solid, hydrophobic, microscopic beads. At the bead/water interface, the copolymers all form stable films in which the nominal molecular areas correspond to those of the molecules when they are packed rather tightly at the air/water interface. Beads coated with condensed films of copolymers that contain short PEO segments and elicit appreciable inflammation absorb appreciable quantities of plasma proteins, including fibrinogen, from aqueous solution. Beads coated with fibrinogen aggregate when they are stirred in the presence of thrombin, a consequence of interbead fibrin formation. Beads coated with condensed films of copolymers that contain long PEO segments and elicit little inflammation absorb little plasma protein, and they do not aggregate in the presence of thrombin. Our data and observations are consistent with the prevailing notion that the utility of triblock copolymers as agents for modifying the surface properties of blood‐contacting surfaces derives from the influence of the copolymers on the adsorption of plasma proteins. In this regard, the ability of the copolymers to influence fibrinogen‐mediated adhesive events may be particularly important. As to the mechanism of protein resistance, our data support the proposal that sibling PEO segments of copolymers in condensed films fold back across their parental PPO cores, limiting access of proteins to the hydrophobic cores themselves.
Scale‐up process for expression and renaturation of recombinant human epidermal growth factor from Escherichia coli inclusion bodies
Authors: Lee Jong Yeon , Yoon Chang Shin , Chung Il Yup , Lee Young Seek , Lee Eun Kyu
Published: November 2003

A cDNA encoding mature epidermal growth factor (EGF) was isolated and cloned into a pQE30 vector in which the His6‐tagged EGF was expressed. pH‐stat feeding of concentrated medium at the time of isopropyl β‐d‐thiogalactoside induction and slug‐feedings of the enriched medium during the induction resulted in a higher cell density and specific expression. Using a simple refolding protocol that consisted of 1 mM l‐cysteine addition for a 1‐h reduction followed by 5 mM l‐cystine addition for oxidative refolding, we were able to convert nearly all EGF monomers into the oxidized form. Also, the refolding aggregate was converted into the monomeric form. Approx. 50% overall yield was obtained from the dissolved inclusion bodies to a single peak under FPLC. We hope that the result of this study may provide information that is useful for the scale‐up of the recombinant human EGF production process.
Conformational issues in the characterization of proteins
Authors: Price Nicholas C.
Published: November 2003

The conformation of a protein refers to the three‐dimensional arrangement of its constituent atoms. Since expression of the biological activity of a protein depends on its conformation, it is clear that full characterization of a protein involves an understanding of its three‐dimensional structure. This review outlines the principal techniques for determining the conformation of a protein, describes the crucial role played by the flexibility of proteins, and gives an account of current theories of the mechanisms by which proteins fold. A final section deals with strategies that can be adopted to preserve the conformational integrity of proteins; an aspect that is of increasing importance as a greater number of proteins are finding applications in industrial processes and as therapeutic and diagnostic agents.
Replacement of methionine‐161 with threonine eliminates a major by‐product of human glutamate decarboxylase 65‐kDa variant expression in Escherichia coli
Authors: Santos Javier , Antón Erica A. , Buslje Cristina Marino‐ , Valdez Silvina N. , Villanueva Ana L. , Sica Mauricio , Iacono Rubén , Maffia Paulo , Poskus Edgardo , Ermácora Mario R.
Published: November 2003

Most insulin‐dependent diabetes mellitus patients gen‐erate conformational autoantibodies to the islet‐cell 65‐kDa variant of human glutamate decarboxylase (GAD65), and several immunochemical tests for the early detection of type‐1 diabetes rely on GAD65 antibody (GADA) assessment using properly folded recombinant GAD65 as the antigen. In addition, preventive therapies based on tolerization by GAD65 administration may be available in the near future. Therefore, there exists a strong interest in a facile and economically sound expression procedure for this antigen. Several attempts to produce, in native form, wild‐type GAD65 in Escherichia coli have failed. However, this difficulty was recently surmounted in our laboratory by expressing GAD65 as a fusion protein with thioredoxin [Papouchado, Valdez, Ghiringhelli, Poskus and Ermácora (1997) Eur. J. Biochem. 246, 350–359]. In this work, a new GAD65 hybrid gene was prepared by joining engineered cDNA obtained from human and rat tissues. The new gene was modified additionally to finally code for human GAD65 with a single amino‐acid substitution: Met‐161→Thr. This change impeded the co‐expression of a 48‐kDa by‐product from an internal translation site. Also, a second 58‐kDa by‐product was identified as a GAD65 C‐terminal proteolytic fragment that co‐purifies with thioredoxin–M161T GAD65. The new GAD65 variant was expressed and easily purified, yielding an antigen that performed equally or better than wild‐type GAD65 in the reference radiobinding assay for GADA. The procedure provides an inexpensive source of large amounts of fully active and immunochemically competent GAD65.
Direct analysis of single‐nucleotide polymorphism on double‐stranded DNA by pyrosequencing
Authors: Nordström Tommy , Ronaghi Mostafa , Forsberg Lena , De Faire Ulf , Morgenstern Ralf , Nyrén Pål
Published: November 2003

Pyrosequencing, a new method for DNA sequencing, is gaining widespread use for many different types of DNA analysis. The method takes advantage of four coupled enzymes in a single tube assay to monitor DNA synthesis in real time using a luminometric detection system. Here, we demonstrate the use of pyrosequencing for direct analysis of single‐nucleotide polymorphism on double‐stranded PCR product. Pyrosequencing data on the human glutathione peroxidase gene (GPX1) from several individuals were analysed and three different allelic variants were determined and confirmed. The possibility of further simplifying the sequencing and template‐preparation steps is discussed.
Carbohydrate structures of soluble human L‐selectin recombinantly expressed in baby‐hamster kidney cells
Authors: Gohlke Martin , Mach Ulrich , Nuck Rolf , Zimmermann‐Kordmann Martin , Grunow Detlef , Fieger Claudia , Volz Barbara , Tauber Rudolf , Petri Thomas , Debus Nils , Reutter Werner
Published: November 2003

A soluble form of L‐selectin was recombinantly produced, which might be an effective therapeutic agent in inflammatory disorders, acting as an inhibitor for leucocyte endothelium adhesion. In the present study the oligosaccharide structures of soluble human L‐selectin, recombinantly expressed in baby‐hamster kidney cells, were determined. The N‐linked glycans were enzymically released and fluorescently labelled with 2‐aminobenzamide. Sialylation of the N‐glycans was analysed by anion‐exchange chromatography followed by rechromatography of the resulting fractions on amino‐phase HPLC after release of the sialic acid residues. Desialylated oligosaccharides were separated using two‐dimensional HPLC and characterized by digestion with exoglycosidases and MS. More than 30 oligosaccharide structures representing at least 95% of the overall glycosylation of this protein were determined. The results revealed that recombinant soluble human L‐selectin carries bi‐, tri‐ and tetra‐antennary sugar chains, which are fucosylated on the innermost residue of N‐acetylglucosamine. The number of sialic acid residues linked to these glycans ranges from 0 (neutral glycans) to 4 (tetrasialylated oligosaccharides). The sialic acid is found exclusively in the α2–3 linkage to galactose. In addition to the main glycans, different minor structures containing terminal N‐acetylgalactosamine, or the H (O) blood‐group determinant were also identified. O‐Glycosylation of mucin‐type sugar chains was not detected in recombinant soluble human L‐selectin.
Purification of native enolase from medically important Candida species
Authors: Ballantyne Denis S. , Warmington John R.
Published: November 2003

The 48 kDa glycolytic enzyme, enolase, has been identified as an immunodominant antigen in Candida albicans infections. It has also been identified as an important fungal allergen. Enolase from a number of medically important Candida species has been purified using a two‐step anion‐ and cation‐exchange chromatography method that was preceded by an organic extraction. The enolases purified by this method have a high specific activity and the procedure is 40% efficient, with an average of 5 mg of enolase/g of Candida cells. The purification of native enolase from medically important Candida species will enable the immunological significance and interspecies relationships of this major fungal antigen to be investigated.
Development of allergen‐coated paper discs for allergy diagnosis by ELISA
Authors: Singh Bhanu Pratap , Sridhara Susheela , Malhotra Mittoo , Tulsani Narayan B. , Gangal Sharad V.
Published: November 2003

In vitro testing is useful for detecting pollen and fungal‐allergen sensitivities in nasobronchial‐allergy patients. However, allergen‐coated discs or solid‐phases supplied in kits do not contain all the relevant extracts. The present study was aimed to develop a safe and credible procedure of allergen coating on paper discs for allergy diagnosis by ELISA. Paper discs were coated with indigenous allergens using poly(vinyl alcohol) and glutaraldehyde. ELISAs were carried out to standardize the allergen‐coated discs using skin‐test‐positive‐patients’ sera. The cut‐off value (A) for ELISA was worked out by analysing normal human sera (NHS) and is based on the mean±2 S.D. for the NHS value. The allergen‐coated discs, i.e. Centre for Biochemical Technology (CBT) discs, demonstrated excellent comparison with plate‐binding method and Pharmacia discs [Prosopis juliflora (mesquite), critical value 0.879 at 5% probability±0.599, n=11, is significant]. Inter‐ and Intra‐assay coefficients of variations ranged from 2.5–14.05% to 4.8–11.5% with different allergens. Results of skin tests and ELISA with CBT discs showed 60–70% correlation.
Purification and characterization of biosurfactants from Nocardia sp. L‐417
Authors: Kim Soon Han , Lim Ee Jong , Lee Sang Ok , Lee Jae Dong , Lee Tae Ho
Published: November 2003

The Nocardia sp. L‐417 strain grown with n‐hexadecane as a carbon source produced two types of biosurfactant that have different characteristics. These biosurfactants were purified by procedures that included ammonium sulphate fractionation, chilled acetone and hexane treatments, silica‐gel column chromatography and Sephadex LH‐20 gel filtration. The purified biosurfactants were very stable over a broad range of pHs (2–12) and temperatures (100 °C, 3 h). The biosurfactant type I had strong properties as an emulsifying agent and as an emulsion‐stabilizing agent, whereas type II had a strong ability to reduce surface tension.
Improvement of Staphylococcus aureus‐V8‐protease hydrolysis of bovine haemoglobin by its adsorption on to a solid phase in the presence of SDS: peptide mapping and obtention of two haemopoietic peptides
Authors: Vercaigne‐Marko Dominique , Kosciarz Elisabeth , Nedjar‐Arroume Naima , Guillochon Didier
Published: November 2003

Hydrolysis of bovine haemoglobin by the V8 protease from Staphylococcus aureus (EC 3.4.21.19) was studied in the presence of SDS in a homogeneous‐phase and in a solid‐phase system. In both cases, hydrolyses were performed at 37 °C, in 50 mM phosphate buffer, pH 6.0, containing 0.1% SDS. Solid‐phase hydrolyses were carried out with haemoglobin adsorbed on a negatively charged hydrophobic support, namely Amberlyst 15Wet (Rohm and Haas). The peptides were isolated from the hydrolysates by reverse‐phase HPLC and analysed for their amino acid composition on a Waters Pico‐Tag column, confirmed by second‐order derivative spectrometry or by MS. A peptide map of the hydrolysates was drawn up, and numerous new cleavages in haemoglobin chains were observed, especially after Asp. This study showed that SDS permitted a dramatic improvement in the hydrolysis of whole haemoglobin by V8 protease in both homogeneous‐phase and solid phase systems after adsorption of haemoglobin on to an anionic support. Moreover, in the heterogeneous phase, all the theoretical cleavage sites of V8 protease, Asp as well as Glu bonds, were hydrolysed, except for four sites which were resistant owing to strong interactions with the support. These results led to us obtain two haemopoietic peptides, namely peptide α (Leu76‐Pro‐Gly‐Ala‐Leu‐Ser‐Glu82) and peptide β (Lys94‐Leu‐His‐Val‐Asp‐Pro‐Glu100). These active peptides have never before been prepared from bovine haemoglobin, and they may have great potentialities in biotechnology.
B‐cell epitope mapping of theNeisseria meningitidisP64k protein using overlapping peptides
Authors: González Sonia , Viña Lisel , Nazábal Consuelo , Chinea Glay , Caballero Evelin , Musacchio Alexis
Published: November 2003

A common meningococcal antigen designated P64k has been identified, cloned and expressed inEscherichia coli. The recombinant antigen is highly immunogenic in several animal species and its immunogenicity in healthy human volunteers is under investigation. Recently, P64k has been used as an immunological carrier for weak immunogens. To characterize the B‐cell epitopes on P64k, recognized by immune sera obtained from mice, rabbits and monkeys, multiple overlapping peptides were synthesized and screened for antibody binding. Peptides covering the complete sequence of the P64k protein, 59 in all, of 20 amino acids each (overlapped by 10 residues), were synthesized. A number of continuous epitopes were detected with all sera, when immune and pre‐immune bleeds were compared. For mouse and monkey sera, a few major antigenic peptides were identified, while the recognition of the rabbit serum was much more heterogeneous. Despite variation in the exact location of continuous epitopes defined by different anti‐P64k sera, we found an immunogenic core region within the molecule, composed of amino acids Asp524–Gly533. Consistently, in this protein segment there was an amino acid stretch located in a β‐hairpin loop, which is exposed to the solvent in the previously determined three‐dimensional structure of the protein. This region is protruding and accessible to a sphere with a radius of 9 Å.
Expression, purification and applications of staphylococcal Protein A fused to cellulose‐binding domain
Authors: Shpigel Etai , Goldlust Arie , Eshel Adi , Ber Idit Kaplan , Efroni Gilat , Singer Yossi , Levy Ilan , Dekel Mara , Shoseyov Oded
Published: November 2003

Because staphylococcal Protein A (ProtA) binds specifically to IgG, it has been used for many immunological manipulations, most notably antibody purification and diagnostics. Immobilization is required for most of these applications. Here we describe a genetic‐engineering approach to immobilizing ProtA on cellulose, by fusing it to cellulose‐binding domain (CBD) derived from the cellulose‐binding Protein A of Clostridium cellulovorans. The bifunctional fusion protein was expressed in Escherichia coli, recovered on a cellulose column and purified by elution at alkaline pH. ProtA–CBD was used to purify IgG from rabbit serum and its ability to bind IgG from different sources was determined. The bifunctional chimaeric protein can bind up to 23.4 mg/ml human IgG at a ratio of 1 mol of ProtA–CBD/2 mol of human IgG, and can purify up to 11.6 mg/ml rabbit IgG from a serum. The ability to bind functionally active CBD‐affinity reagents to cellulosic microtitre plates was demonstrated. Our results indicate that a combination of CBD‐affinity reagents and cellulosic microtitre plates is an attractive diagnostics matrix for the following reasons: (i) cellulose exhibits very low non‐specific binding; and (ii) CBD‐fusion proteins bind directly to cellulose at high density. A unique signal‐amplification method was developed based on the ability of ProtA–CBD to link stained cellulose particles to primary antibody in a Western blot.
Stabilizing effect of chemical additives against oxidation of lactate dehydrogenase
Authors: Andersson Maria M. , Breccia Javier D. , Hatti‐Kaul Rajni
Published: November 2003

Oxidation is one of the major pathways for denaturation of proteins during storage, and also a potential problem in protein production, isolation and purification processes. In this study, a number of additives have been tested for their protective effects against oxidation in the presence of metal ions and hydrogen peroxide. Porcine muscle lactate dehydrogenase (LDH) was used as a model protein. Oxidation and denaturation of the enzyme were followed as activity loss, modification of certain amino acids, altered secondary structure and aggregation. Loss of activity during metal‐catalysed oxidation was accompanied by structural damage of the enzyme, which was not the case during oxidation with peroxide. The best protectant during both modes of oxidation was found to be the polycation, poly(ethyleneimine) (PEI), followed by EDTA. Both chemicals also increased the enzyme's half‐life during oxidation with a mixture of copper ions and hydrogen peroxide. Ammonium sulphate was an effective stabilizer during metal‐catalysed oxidation, while sorbitol, sucrose and hydroxyectoine provided moderate stabilization. The ectoine was also stabilizing against oxidation with hydrogen peroxide, as was poly(ethylene glycol), whereas sorbitol enhanced the rate of enzyme inactivation. The stability of LDH towards denaturation by both oxidation and temperature was increased by addition of both PEI and sorbitol, as indicated by the melting‐temperature profiles of the enzyme.
Purification of alkaline phosphatase from chicken intestine by expanded‐bed affinity chromatography on dye‐linked cellulose
Authors: Roy Ipsita , Gupta Munishwar N.
Published: November 2003

Alkaline phosphatase from chicken intestine was purified on dye‐linked cellulose beads. The various dye‐linked cellulose beads were screened in the batch mode for purification of the enzyme and the promising dyes were tried in the expanded‐bed mode. The one‐step expanded‐bed affinity chromatography on Cibacron Blue‐linked cellulose beads yielded 48‐fold purification with an activity recovery of 70%.
Mechanisms of protein modification during model anti‐viral heat‐treatment bioprocessing of β‐lactoglobulin variant A in the presence of sucrose
Authors: Smales C. Mark , Pepper Duncan S. , James David C.
Published: November 2003

To ensure the safety of plasma and recombinant therapeutic proteins, heat treatment is routinely applied to these biopharmaceuticals as a means of virus inactivation. However, to maintain protein integrity during heat treatment it is necessary to use high concentrations of thermostabilizing excipients, such as sucrose, in order to prevent protein damage. In this study we describe the covalent modifications inferred to a model protein, β‐lactoglobulin A, that occur during typical and extended anti‐viral heat treatments. The chemical derivation and mechanisms by which these modifications arise are addressed. Heat treatment initiated hydrolysis of sucrose to glucose and fructose, which in turn were degraded to glyoxal. Glyoxal and the free reducing sugars reacted with free amino groups in β‐lactoglobulin A to yield Maillard glycation adducts and advanced glycation end products (AGEs). The major mechanism for AGE formation was via degradation of glucose‐derived Schiff‐base adducts. Heat treatment and glycation of β‐lactoglobulin A resulted in thiol‐disulphide interchange reactions leading to protein oligomerization. A small population of β‐lactoglobulin A non‐disulphide‐linked dimers were also observed with increasingly harsh heat treatments. These findings have implications for (i) improvements in the safety and efficacy of heat‐treated protein biopharmaceuticals and (ii) our understanding of the mechanisms of protein glycation and AGE adduct formation.
Design and production of recombinant subunit vaccines
Authors: Hansson Marianne , Nygren Per‐A˚ke , Sta˚hl Stefan
Published: November 2003

The development of subunit vaccines is presently the main strategy being evaluated for prevention of infectious diseases. The use of recombinant‐DNA techniques has facilitated the development of new principles for design and production of subunit vaccines. First of all, the properties of a target protein immunogen can be improved by the use of gene‐fusion technology or by the creation of specific changes, to generate ‘second‐generation protein vaccines’. Properties that can be modified include protein solubility, protein stability, in vivo half‐lives, etc. In addition, for subunit protein vaccine candidates, the immunogenic properties can be significantly augmented by the addition of immunopotentiating tags or by means of targeting to immunoreactive sites. The recombinant subunit vaccine can furthermore be adapted by gene‐fusion technology, to be efficiently incorporated into immunopotentiating adjuvant systems. Also in passive vaccination strategies, i.e. the use of antibodies or antibody fragments for prevention of infectious diseases, the recombinant strategies have become increasingly important. Humanized antibodies and antibody fusion proteins represent common present anti‐infectious‐disease agents. The selected examples will indicate that recombinant strategies will indeed have an impact on the design, selection and production of recombinant proteins to be used in the prevention of infectious diseases.
Optimization of 6‐aminopenicillanic acid (6‐APA) production by using a new immobilized penicillin acylase
Authors: Torres‐Bacete Jesús , Arroyo Miguel , Torres‐Guzmán Raquel , De La Mata Isabel , Castillón María Pilar , Acebal Carmen
Published: November 2003

A new immobilized penicillin acylase (ECPVA) was obtained by covalent binding of penicillin acylase from Streptomyces lavendulae on Eupergit C. Enzymic hydrolysis of penicillin V catalysed by ECPVA was optimized using a 23 factorial design of experiments, and the selected parameters for this study were pH, temperature and substrate concentration. The immobilized enzyme showed an optimal pH value of 9.5–10.5, and an optimal temperature of 60 °C, whereas its soluble counterpart showed the same optimal pH value and a lower optimal temperature of 50 °C.
Biomedical implications of protein folding and misfolding
Authors: Sadana Ajit , Vo‐Dinh Tuan
Published: November 2003

A review of protein‐folding mechanisms is presented to indicate (i) the formation of correctly folded and misfolded forms, and (ii) the biomedical implications involved. Protein mechanisms may be classified into series and series‐parallel mechanisms. The mechanism may involve one or more stable intermediate (partially folded, misfolded, with some structure) states. It is the parallel or the ‘off‐pathway’ step(s) that lead to the aggregate (misfolded state). This aggregate state yields the amyloid deposits in human tissue that lead to diseases in humans that are resistant to treatment. Means by which the off‐pathway step may be minimized and the correct folding pathway enhanced are presented. Particular emphasis is placed on thein vivomachinery (chaperones) that is in place in the cells, and how it functions to allow the proteins to fold in the correct and active form, thus minimizing the amyloid deposits in tissues. More effort needs to be placed to understand the function of these chaperones, and thus help facilitate the formation of active and stable proteins in the cells. Though more effort needs to be placed in the present treatment strategies, novel treatment procedures also need to be explored in order to develop effective strategies to treat these seemingly intractable diseases.
Rabies virus production in non‐woven polyester fabric (NWPF) packed‐bed reactors
Authors: Gümüşderelioğlu Menemşe , Aslankaraoğlu Esin , Gürhan S. İsmet
Published: November 2003

The production of rabies virus from baby hamster kidney‐Ankara 66 (BHK‐An66, HUKUK 99050302) monolayer cells was examined in a packed‐bed reactor containing non‐woven polyester fabric (NWPF) discs. At first, growth characteristics of the cells were determined in static culture. The suspension culture studies were realized in a 1 litre spinner basket with NWPF support. BHK‐An66 cells (inoculation density, 5×104 cells·ml−1) were maintained in the reactor loaded with 5 g·l−1 carrier. The culture medium was Eagle's minimal essential medium supplemented with 10% (v/v) fetal bovine serum at 37 °C. During the culture, the medium was sampled daily to assess glucose and lactate concentrations. At the end of the 7 day culture period the cell density was found to be 2.2×107 cells·ml−1, and the reactor was inoculated with 5 ml [1.7×106 focus‐forming units (ffu)·ml−1] of the CVS 11 (Challenge Virus Standard) strain of rabies virus. After a 72 h incubation period, the cultures were stained with fluorescein‐conjugated anti‐rabies globulin and were observed using a fluorescence microscope. Virus titres determined by the Spearman–Kärber method were 2.2×105 ffu·ml−1. In conclusion, NWPF packed reactors can be considered as a suitable system for the large‐scale production of rabies virus.
Preparation and characterization of a new bioinorganic composite: collagen and hydroxyapatite
Authors: Mythili J. , Sastry T. P. , Subramanian M.
Published: November 2003

Studies on biomineralization in bone have opened new arenas in the field of fabrication and engineering of bone‐graft devices. A new bioinorganic composite was prepared by introducing processed collagenous matrices into the calcifying solutions at pH 7.8–8.0. The calcifying solutions were prepared by using a saturated dicalcium salt solution. Hydroxyapatite grew on the matrix over a period of 3–5 days in a specially designed crystallizing chamber. Changes in biochemical characters and biophysical parameters have given interesting insights into factors affecting and effecting initiation and progression of calcification on organic matrices.
Improved recovery of insulin‐like growth factors (IGFs) from bovine colostrum using alkaline diafiltration
Authors: Hossner Kim L. , Yemm Robert S.
Published: November 2003

Studies to develop a rapid, bioprocess‐compatible method to recover low‐molecular‐mass growth factors from bovine colostrum are reported. Defatted bovine colostrum was subjected to tangential‐flow filtration (TFF) using two different filters [polyether sulphone (PES) and regenerated cellulose (RC)] at pH 5.8, pH 8.0 and pH 8.0+0.01 M NaCl. Single‐pass TFF at pH 5.8 using a 100 kDa RC filter resulted in the loss of approx. 90% of insulin‐like growth factor I (IGF‐I) to non‐specific filter adsorption. Comparison of 30 kDa RC and PES filters under single‐pass conditions showed that yields of IGF‐1 and IGF‐II were highest with RC filters. Yields of IGF‐I and protein from both filter types were increased at pH 8.0 and were greatest for the 30 kDa RC filter. Effects of adding large diluent volumes continuously during TFF (diafiltration) were tested at pH 5.0 and 8.0. The use of 10 diafiltrate vols. at pH 8.0 resulted in the recovery of 15–28% of colostral IGF‐1 from the RC 30 kDa permeates, 2–4‐fold greater than under acidic conditions. Yields of IGF‐II (39.6%) were unaffected by pH and at least 97% of total protein was retained by the 30 kDa filter at pH 8.0. Denaturing SDS/PAGE analysis of the alkaline RC 30 kDa permeates demonstrated two major regions of stained proteins at 10–13 kDa and 17–19 kDa. Acidic TFF permeates contained additional stained proteins at≈90, 48 and 37 kDa. Isoelectric focusing of these samples demonstrated the presence of proteins with isoelectric points of 8.2 and 8.6. The current study demonstrates a one‐step bioprocess‐compatible technique for the recovery of low‐molecular‐mass polypeptides from bovine colostrum. By using alkaline diafiltration with RC filters TFF provided optimal recovery of IGF‐1 from colostrum.
Efficiency of promoter and cell line in high‐level expression of erythropoietin
Authors: Park Jang Hyeon , Kim Chun , Kim Won Bae , Kim Young Kook , Lee Se Young , Yang Jai Myung
Published: November 2003

Efficiency of viral promoters and various cell lines in directing high‐level expression of human erythropoietin (Epo) was investigated. To investigate the effects of various viral promoters and cell lines on the Epo expression level, genomic Epo with the 5′ and 3′ untranslated regions (UTRs) deleted was cloned next to the simian virus 40 early promoter, cytomegalovirus early promoter or SRα promoter. These expression vectors were transfected into COS‐7, BHK‐21 and Chinese hamster ovary (CHO)/dhfr−cells, respectively. The COS‐7 cells transfected with the vector containing the SRα promoter showed the highest expression level (≈103 IU/ml) at 72 h post‐transfection. For the development of Epo‐producing stable cell lines, BHK‐21 and CHO/dhfr−cells transfected with the 5′,3′‐UTR‐deleted genomic Epo under the control of the SRα promoter were cultured with media containing zeocin. Several clones of zeocin‐resistant BHK‐21 and CHO/dhfr−cells were cultured in the presence of methotrexate (MTX). A BHK‐21 clone selected in the presence of 500 nM MTX expressed and secreted ≈490 IU/ml Epo into the medium. A CHO/dhfr−clone selected in the presence of 20 nM MTX expressed and secreted ≈45 IU/ml Epo into the medium. Southern‐blot analysis indicated that enhancement of Epo expression in the MTX‐resistant stable cells might be related to the amplification of gene copy number.
Site‐specific and random immobilization of thermolysin‐like proteases reflected in the thermal inactivation kinetics
Authors: Mansfeld Johanna , Ulbrich‐Hofmann Renate
Published: November 2003

Immobilization of proteins usually leads to random orientation of the molecules on the surface of the carrier material, whereby mechanistic interpretations of changes in properties, such as thermal stability, become very difficult. Recently, we have prepared several mutant enzymes of the thermolysin‐like neutral protease from Bacillus stearothermophilus, containing cysteine residues in different positions on the surface of the protein molecule. These enzymes allowed site‐specific immobilization to Activated Thiol‐Sepharose and showed that stabilization effects strongly depend on the position of attachment [Mansfeld, Vriend, Van den Burg, Eijsink and Ulbrich‐Hofmann (1999) Biochemistry 38, 8240–8245]. The greatest stabilization was achieved after immobilization of the mutant enzymes S65C and T56C/S65C within the structural region (positions 56–69) where unfolding is initiated. In this study thermal inactivation kinetics of these two mutant enzymes, as well as those of the pseudo‐wild‐type enzyme and thermolysin, were compared for different types of immobilization. Besides site‐specific immobilization via thiol groups, the enzymes were bound randomly via their amino groups or by mixed‐type binding. The basic matrix was Sepharose 4B in all carriers. Whereas the enzymes bound site‐specifically to Activated Thiol‐Sepharose showed clear first‐order inactivation kinetics like the soluble enzymes, the other immobilized enzyme preparations were characterized by distinct biphasic inactivation kinetics reflecting the heterogeneity of enzyme molecules on the carrier with respect to thermal unfolding. Site‐specific binding resulted in stronger stabilization than the mixed binding type. However, immobilization to a highly functionalized carrier via amino groups increased stability further, suggesting that multiple fixation outside of the unfolding region 56–65 is able to increase stability of the enzyme molecules additionally.
An Aspergillus chitosanase with potential for large‐scale preparation of chitosan oligosaccharides
Authors: Cheng Chih Yu , Li Yaw‐Kuen
Published: November 2003

A chitosan‐degrading fungus, designated Aspergillus sp. Y2K, was isolated from soil. The micro‐organism was used for producing chitosanase (EC 3.2.1.132) in a minimal medium containing chitosan as the sole carbon source. The induced chitosanase was purified to homogeneity from the culture filtrate by concentration and cationic SP‐Sepharose chromatography. The purified enzyme is a monomer with an estimated molecular mass of 25 kDa by SDS/PAGE and of 22 kDa by gel‐filtration chromatography. pI, optimum pH and optimum temperature values were 8.4, 6.5 and 65–70 °C, respectively. The chitosanase is stable in the pH range from 4 to 7.5 at 55 °C. Higher deacetylated chitosan is a better substrate. Chitin, xylan, 6‐O ‐sulphated chitosan and O ‐carboxymethyl chitin were indigestible by the purified enzyme. By endo‐splitting activity, the chitosanase hydrolysed chitosan to form chitosan oligomers with chitotriose, chitotetraose and chitopentaose as the major products. The enzyme hydrolyses chitohexaose to form chitotriose, while the chitopentaose and shorter oligomers remain intact. The N‐terminal amino acid sequence of the enzyme was determined as YNLPNNLKQIYDDHK, which provides useful information for further gene cloning of this enzyme. A 275 g‐scale hydrolysis of chitosan was performed. The product distribution was virtually identical to that of the small‐scale reaction. Owing to the simple purification process and high stability of the enzyme, it is potentially valuable for industrial applications.
Anthrax‐toxin‐mediated delivery of a 19 kDa antigen of Mycobacterium tuberculosis into the cytosol of mammalian cells
Authors: Mehra Varsha , Khanna Hemant , Chandra Ramesh , Singh Yogendra
Published: November 2003

PA63, the proteolytically activated 63 kDa fragment of protective antigen (PA, 83 kDa), mediates translocation of lethal factor (LF) and oedema factor into the cytosol. The N‐terminal 254 amino acids of LF (LFn) are required for binding to PA63 and mediating translocation of active ligands fused to either the N‐ or C‐terminus. Here we report translocation of a 19 kDa antigen of Mycobacterium tuberculosis into the cytosol of mammalian cells when fused to the C‐terminus of LFn (LFn‐19kDa). The fusion protein was non‐toxic to J774A.1 macrophage cells in combination with PA and retained the ability to bind to PA63 when incubated with Chinese hamster ovary K1 cells. The data show the efficacy of anthrax toxin to mediate translocation of M. tuberculosis antigens into the cytosol of mammalian cells and may prove useful in delivering proteins and peptides carrying immunodominant mycobacterial antigens into the cytosol.
Improved preparation and crystallization of 25 kDa human fibroblast growth factor‐9
Authors: Koyama Nobuyuki , Ohmae Hiroaki , Tsuji Shinji , Tanaka Yoko , Kurokawa Tsutomu , Nishimura Osamu
Published: November 2003

We prepared 25 kDa human fibroblast growth factor‐9 (hFGF‐9 N33) on a large scale after overproduction in Escherichia coli MM294 (DE3)/pTG931. The purification was performed by a combination of hydrophobic chromatography and HPLC with an ion exchange column, a heparin affinity column and a gel filtration column. This improved procedure was rapid and simple, and the purified hFGF‐9 N33 was found to be homogeneous as judged by various criteria, such as amino acid analysis, N‐terminal amino acid sequence, C‐terminal amino acid analysis and biological activity. Furthermore, as determined by low endotoxin and DNA content, the protein was of high purity. In addition, the hFGF‐9 N33 prepared in the present study was easily crystallized by the vapour diffusion method.