Analytical and preparative native polyacrylamide gel electrophoresis: Investigation of the recombinant and natural major grass pollen allergen Phl p 2

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot are amongst the most popular methods for allergen characterization, such as comparison of recombinant allergens with their natural counterparts. Native PAGE was evaluated as a possible robust and simple method offering high-resolution capacity for characterization of the major grass pollen allergen Phl p 2. Analytical separation of recombinant Phl p 2 provided a superior quality control in terms of homogeneity and, after Western blotting, immunoglobulin E (IgE) reactivity. Separation of natural Phl p 2 identified two major isoforms which were shown to have different N-terminal sequences and IgE-binding properties. After isolation using preparative native PAGE in combination with electrodialysis, both isoforms were investigated by specific proteolysis and reversed-phase high-performance liquid chromatography (RP-HPLC). The results demonstrate differences in the primary structures and that the recombinant counterpart corresponds exactly to one isoform. Analytical and preparative native PAGE thus proved to be powerful tools for the investigation of allergen isoforms and quality control of recombinant counterparts.     

Co-overexpression of PpPDI Enhances Secretion of Ancrod in Pichia pastoris

Ancrod, a serine protease purified from the venom of Agkistrodon rhodostoma, is highly specific for fibrinogen. It causes anticoagulation by defibrinogenation and has been used as a therapeutic anticoagulant for the treatment of moderate to severe forms of peripheral arterial circulatory disorders in a variety of countries. The DNA of ancrod was amplified by recursive PCR with a yeast bias codon and cloned into the pGEM-T Easy vector. In order to achieve a high level secretion and a full activity expression of ancrod in Pichia pastoris (P. pastoris), the P. pastoris protein disulfide bond isomerase (PpPDI) was co-overexpressed in the strain. The secretion characteristics of ancrod with and without PpPDI were examined. With co-overexpression of PpPDI, the production of recombinant ancrod (rAncrod) was increased to 315 mg/L in the culture medium, which is twofold higher than the control strain carrying only the ancrod gene. Through purified by Ni2? affinity chromatography and phenyl Sepharose column, the purity of rAncrod was found to be as high as 95.2%. The fibrinogenolytic and zymographic activities of the rAncrod were determined and found to be similar to that of the native protein. This improved expression system can facilitate further studies and the industrial production of ancrod.     

N-Terminomics Strategies for Protease Substrates Profiling

Proteases play a central role in various biochemical pathways catalyzing and regulating key biological events. Proteases catalyze an irreversible post-translational modification called proteolysis by hydrolyzing peptide bonds in proteins. Given the destructive potential of proteolysis, protease activity is tightly regulated. Dysregulation of protease activity has been reported in numerous disease conditions, including cancers, neurodegenerative diseases, inflammatory conditions, cardiovascular diseases, and viral infections. The proteolytic profile of a cell, tissue, or organ is governed by protease activation, activity, and substrate specificity. Thus, identifying protease substrates and proteolytic events under physiological conditions can provide crucial information about how the change in protease regulation can alter the cellular proteolytic landscape. In recent years, mass spectrometry-based techniques called N-terminomics have become instrumental in identifying protease substrates from complex biological mixtures. N-terminomics employs the labeling and enrichment of native and neo-N-termini peptides, generated upon proteolysis followed by mass spectrometry analysis allowing protease substrate profiling directly from biological samples. In this review, we provide a brief overview of N-terminomics techniques, focusing on their strengths, weaknesses, limitations, and providing specific examples where they were successfully employed to identify protease substrates in vivo and under physiological conditions. In addition, we explore the current trends in the protease field and the potential for future developments.     

Production of poplar xyloglucan endotransglycosylase using the methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The gene XET16A encoding the enzyme xyloglucan endotransglycosylase (XET) from hybrid aspen (Populus tremula x tremuloides Mich) was transformed into Pichia pastoris GS115 and the enzyme was secreted to the medium. The influence of process conditions on the XET production, activity, and proteolytic degradation were examined. Inactivation of XET occurred in the foam, but could be decreased significantly by using an efficient antifoam. Rich medium (yeast extract plus peptone) was needed for product accumulation, but not for growth. The proteolytic degradation of the enzyme in the medium was substantially decreased by also adding yeast extract and peptone to the glycerol medium before induction with methanol. Decreasing the fermentation pH from 5.0 to 4.0 further reduced the proteolysis. The specific activity was further improved by production at 15 degrees C instead of 22 degrees C. In this way a XET production of 54 mg/L active enzyme could be achieved in the process with a specific activity of 18 Unit/mg protein after a downstream process including centrifugation, micro- and ultrafiltration, and ion exchange chromatography.     

Culture conditions dictate protease and tannase production in submerged and solid-state cultures of Aspergillus niger Aa-20

Undesirable protease production by Aspergillus niger Aa-20 in submerged culture and solid-state culture was evaluated using different concentrations of tannic acid as sole carbon source in a model system designed for tannase production. Protease production was found to be dependent on the culture system used (submerged culture or solid-state culture) and on the initial tannic acid concentration. Expression of protease activity in submerged culture was higher (up to 10 times) than activity obtained in solid-state culture, using identical culture medium composition. In submerged culture, the lowest final protease activity (0.13 IU) was obtained with the highest tannic acid concentration, while in solid-state culture protease activity was not affected by changes in initial substrate concentration. Absence of detectable proteolytic activity in solid-state culture is related to high production of tannase enzyme. Hence, the use of solid-state culture for fungal enzyme production may allow for higher and more stable enzyme titers present in culture extracts.     

Effects of carbon source on expression of alcohol oxidase activity and on morphologic pattern of YR-1 strain, a filamentous fungus isolated from petroleum-contaminated soils

Soluble alcohol oxidase (AO) activity was detected in the supernatant fraction of a high-speed centrifugation procedure after ballistic cellular homogenization to break the mycelium from a filamentous fungus strain named YR-1, isolated from petroleum-contaminated soils. AO activity from aerobically grown mycelium was detected in growth media containing different carbon sources, including alcohols and hydrocarbons but not in glucose. In previous work, zymogram analysis conducted with crude extracts from aerobic mycelium of YR-1 strain indicated the existence of two AO enzymes originally named AO-1 and AO-2. In the present study, we were able to separate the AO-1 band into two bands depending on culture conditions, carbon source, and polyacrylamide gel electrophoresis (PAGE) separation conditions; the enzyme activity pattern in zymograms from cell-free extracts exhibited three different bands after native PAGE. New nomenclature was used for upper bands AO-1 and AO-2 and lower band AO-3, respectively. The expression of AO activity was studied in the absence of glucose in the culture media and in the presence of hydrocarbons or petroleum as sole carbon source, suggesting that AO expression could be subjected to two regulatory possibilities: carbon catabolite regulation by glucose and induction by hydrocarbons. The possibility of catabolic inhibition of AO by glucose in the active enzyme was also tested, and the results confirm that this kind of regulatory mechanism is not present in AO activity.     

Increased sensitivity for Coomassie staining of sodium dodecyl sulfate-polyacrylamide gels using PhastSystem Development Unit

Staining of proteins in PhastGel gradient media with Coomassie Blue R 350 was considerably improved using a lower concentration of methanol (10% v/v) and 2% ammonium sulfate in the staining solution and 10% acetic acid for destaining. The detection limit in sodium dodecyl sulfate-polyacrylamide gels was lowered by a factor of 10 to about 2 ng per protein band. The Coomassie staining method was adapted to the newly developed silver staining procedure so that both can be used in parallel in PhastSystem.     

Improved detection of lymphocyte membrane proteins in purified form and as a crude mixture using native and denaturing polyacrylamide gel electrophoresis by optimisation of coomassie brilliant blue and silver staining.

Optimised silver staining protocols were devised for the detection of membrane proteins in purified form and as a crude mixture. These were adduced in both sodium dodecyl sulphate (SDS) and native polyacrylamide gel electrophoresis and consisted of ethanol-acetic acid-formaldehyde fixation, Coomassie Brilliant Blue prestaining, Rapidfix pretreatment, formaldehyde enhancement and finally ammoniacal silver staining. With these modifications, numerous staining problems of membrane proteins were overcome. These included reduction in background staining, enhanced detection sensitivity in native gels, elimination of negative staining and the avoidance of metallic silver deposition on the gel surface. In overcoming these problems, some factors determining the colour and stainability of membrane proteins in their native state were determined. Both the anionic Coomassie Brilliant Blue dye and SDS detergent improved the sensitivity of silver staining in native gels, and ammoniacal silver was more sensitive than neutral silver, suggesting silver staining to be a charge dependent process.     

Submerged culture screening of two strains ofStreptomyces sp. with high keratinolytic activity

Keratinases can be used for the production of potentially important hydrolyzed proteins and chemicals. This study investigated the keratinolytic activity ofStreptomyces sp on keratinaceous materials like wool. High levels of proteolytic and keratinolytic activity were obtained after 96 h of culture when two Streptomyces sp strains were grown on basal medium containing mineral salts and 3% (w/v) of defatted wool as a source of energy, carbon, and nitrogen. The cell-free culture filtrates exhibited rapid proteolytic digestion of keratin powder. Currently, the authors are testing whether the enzymatic activity obtained is in fact keratinolytic, and not only an alkaline protease activity.     

Using coomassie blue to stabilize H2O2-guaiacol stained peroxidases on polyacrylamide gels.

Shimoni and Reuveni [1] have reported a procedure which uses Coomassie Brilliant Blue R-250 staining and destaining to stabilize H2O2-guaiacol stained peroxidases on polyacrylamide gels and at the same time detects nonspecific protein bands. The procedure has the advantage that the otherwise orange and unstable peroxidase bands are visualized as blue bands and it, therefore, facilitates laser densitometry of electrophoretically separated peroxidases. In the modification of the procedure reported here, the Coomassie staining reagent was adjusted and the destaining step was eliminated. The modified procedure has added advantages: it simplifies the procedure, increases the consistency of the results across gels, prevents or reduces the staining of nonspecific proteins, and still gives excellent resolution of stable bands which can be used to quantify peroxidase activity.     

Designing and monitoring large-scale processes for biomolecule purification with an automated electrophoresis/staining system

An automated electrophoresis/staining system is advantageous in the design, evaluation and documentation of biomolecule purification processes. Its use from laboratory scale to industrial scale is demonstrated in three major purification processes: (i) superoxide dismutase production (processing 3.51 of a clarified yeast lysate), (ii) staphylococcal enterotoxin B production (processing 440 l of supernatant liquid from a cell culture), and (iii) monoclonal antibody production (processing 143 l of a supernatant liquid from a cell culture). The speed and convenience of the system provide advantages in process monitoring; automated silver staining (60 min) enabled the detection of <0.5 ng μl^?1 contaminating protein.     

Extracellular proteolytic processing ofAspergillus awamori GAI into GAII is supported by physico-chemical evidence

The proportion of glucoamylases, GAI and GAII, in the culture supernatant ofAspergillus awamori fermentations depends on the medium C/N ratio in such a way that the transformation of GAI into GAII is favored by the existence of a surplus of the carbon source in the growth medium. This condition also favors the appearance of the proteolytic activity. The authors report the observation that the shift in the isoenzyme proportion was concomitant to the peak of proteolytic activity. A peptide that may have resulted from the continuous degradation of the GAI C-terminal peptide, Gp-1, was isolated by gel filtration and purified by reversephase chromatography. This peptide matched with the region G¹⁴-A³⁴ of the substrate-binding domain of GAI, thus reinforcing the hypothesis of the extracellular proteolytic processing of GAI.     

Characterisation of protease activity in extracellular products secreted by Giardia duodenalis trophozoites treated with propolis

Results from our laboratory revealed propolis activity on Giardia trophozoites proliferation. Since therapeutic agents can inhibit the activity of proteases related to relevant biologic and physiologic processes of parasites, this study was undertaken to characterise the proteolytic activity of excretory/secretory products (ESP) of trophozoites treated with propolis. ESP was obtained from culture supernatants of trophozoites exposed to 250 and 500 μg mL(-1) of propolis. ESP were tested in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the protein profiles and the protease activity was assayed in gelatin-containing gels. Synthetic inhibitors were used to characterise the protease classes. Treated and non-treated ESP showed a similar protein and hydrolysis pattern. A simple pattern of protein composed by five evident bands of approximately 167, 132, 79, 61 and 51 kDa was found, and the zymograms comprised hydrolysis zones distributed from >170 to 23 kDa. No inhibition was seen on protease activity of propolis-treated trophozoites, whose hydrolysis pattern was similar to control. One may conclude that both ESP degraded gelatin and the activity was predominantly due to cysteine proteases. Although propolis had no effect on the proteolytic activity, further studies could identify the active constituents responsible for propolis antigiardial activity and their mechanisms of action.     

Determination of protein spots separated by two-dimensional polyacrylamide gel electrophoresis

A simple method for quantitating proteins in the spots on two-dimensional polyacrylamide gel electropherograms is described. The system consists in three steps: (1) O'Farrell's two-dimensional gel electrophoresis of the proteins to be analysed; (2) staining of the gels with Coomassie brilliant blue; and (3) determination of the area and integrated density of the stained spots by the Joyce Loebl Magiscan-1 image analysis system. The method can be used for the determination of proteins in the range 0.5-100 micrograms/cm2; the amount of protein involved in most spots detected by the staining method actually falls within this range. As the minimum spot diameter that can easily be handled by the method is about 2 mm, as much as 30 ng of protein in such a spot can be determined. The method can also be applied to autoradiograms.     

Cloning and Characterization of a Novel Aspartic Protease Gene from Marine-Derived <Emphasis Type="Italic">Metschnikowia reukaufii</Emphasis> and its Expression in <Emphasis Type="Italic">E. coli</Emphasis>

Metschnikowia reukaufii W6b isolated from marine environment was found to produce a cell-bound acid protease. The full-length cDNA (cDNASAP6 gene) of the acid protease (SAP6) from the marine-derived yeast M. reukaufii W6b was cloned. The insert was 1,755-bp long and contained an open reading frame of 1,527-bp encoding 508 amino acids. The deduced amino acid sequence included a signal peptide of 16 amino acids. The consensus motifs contained a VLLDTGSSDLRM active site and an ALLDSGTTITQF active site. The protein sequence deduced from the cDNASAP6 gene exhibited 12.9% overall identity with Cwp1 of Saccharomyces cerevisiae and a hydropathy profile characteristic of glycosylphosphatidylinositol cell-wall proteins. The cDNASAP6 gene without 48 bp encoding the signal peptide sequence was subcloned into an expression plasmid pET-24a (+) and fused with a 6-His Tag and transformed into Escherichia coli BL21 (DE3) for recombinant expression of the protease. The expressed fusion protein was found to have a unique band with molecular mass of about 54 kDa. The crude acid protease of the culture of the marine yeast strain W6b and the crude recombinant acid protease had milk clotting activity.     

Protease Probes that Enable Excimer Signaling upon Scission

Peptide‐based probes that fluoresce upon proteolytic cleavage are invaluable tools for monitoring protease activity. The read‐out of protease activity through pyrene excimer signaling would be a valuable asset because the large Stokes shift and the long lifetime of the excimer emission facilitate measurements in autofluorescent media such as blood serum. However, proteolytic cleavage abolishes rather than installs the proximity relationships required for excimer signaling. Herein, we introduce a new probe architecture to enable the switching on of pyrene excimer emission upon proteolytic scission. The method relies on hairpin‐structured peptide nucleic acid (PNA)/peptide hybrids with pyrene units and anthraquinone‐based quencher residues positioned in a zipper‐like arrangement within the PNA stem. The excimer hairpin peptide beacons afforded up to a 50‐fold enhancement of the pyrene excimer emission. Time‐resolved measurements allowed the detection of matrix metalloprotease 7 in human blood serum.     

口蹄疫病毒3ABC基因截短体在毕赤酵母中的表达及鉴定

将长为525 bp的口蹄疫病毒3ABC基因截短体克隆到毕赤酵母表达载体pPIC9K中, 构建了重组表达质粒pPIC9K-3ABCt. 用BglⅡ线性化后, 电转化毕赤酵母菌GS115, 经表型筛选, PCR鉴定, 获得阳性重组菌(GS115/pPIC9K-3ABCt). 然后进行诱导表达, 通过SDS-PAGE和Western blot鉴定表达产物. 结果表明, 重组菌株成功分泌表达了分子量为40000, 具有免疫反应活性, 且呈二聚体形式的目的蛋白. 在96 h时表达量达到最高峰, 占分泌总蛋白的18%, 达到23.4 mg/L. 为进一步研制口蹄疫免疫和感染动物鉴别诊断试剂奠定了基础.     

Enzyme Production by Solid Substrate Fermentation of <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> and <Emphasis Type="Italic">Trametes versicolor</Emphasis> on Tomato Pomace

A process of solid state fermentation (SSF) on tomato pomace was developed with the white-rot fungi Pleurotus ostreatus and Trametes versicolor, using sorghum stalks as support. Operative parameters (humidity, water activity, and size of substrate particles) guaranteeing a good colonization of tomato pomace by both fungi were defined and conditions for production at high titers of the industrially relevant enzymes laccase, xylanase and protease were identified. Significant laccase activity levels (up to 36 U g⁻¹ dry matter) were achieved without any optimization of culture conditions, neither by nutrient addition nor by O₂ enrichment. Furthermore, protease activity levels up to 34,000 U g⁻¹ dry matter were achieved, being higher than those reported for the fungi typically considered as the best protease producers such as Aspergillus strains. Moreover, as one of the most significant results of this study, analysis of P. ostreatus tomato SSF samples by zymogram revealed two bands with laccase activity which had not been detected so far.     

A Chemical Inhibitor of the Skp2/p300 Interaction that Promotes p53‐Mediated Apoptosis

Skp2 is thought to have two critical roles in tumorigenesis. As part of the SCF^Skp2 ubiquitin ligase, Skp2 drives the cell cycle by mediating the degradation of cell cycle proteins. Besides the proteolytic activity, Skp2 also blocks p53‐mediated apoptosis by outcompeting p53 for binding p300. Herein, we exploit the Skp2/p300 interaction as a new target for Skp2 inhibition. An affinity‐based high‐throughput screen of a combinatorial cyclic peptoid library identified an inhibitor that binds to Skp2 and interferes with the Skp2/p300 interaction. We show that antagonism of the Skp2/p300 interaction by the inhibitor leads to p300‐mediated p53 acetylation, resulting in p53‐mediated apoptosis in cancer cells, without affecting Skp2 proteolytic activity. Our results suggest that inhibition of the Skp2/p300 interaction has a great potential as a new anticancer strategy, and our Skp2 inhibitor can be developed as a chemical probe to delineate Skp2 non‐proteolytic function in tumorigenesis.