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Currently, the lack of reliable strategies for the diagnosis and treatment of cancer makes the identification and characterization of new therapeutic targets a pressing matter. Several studies have proposed the Six Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) as a promising therapeutic target for prostate cancer. Although structural and functional studies may provide deeper insights on the role of STEAP1 in cancer, such techniques require high amounts of purified protein through biotechnological processes. Based on the results presented, this work proposes the application, for the first time, of a fed-batch profile to improve STEAP1 biosynthesis in mini-bioreactor Komagataella pastoris X-33 Mut+ methanol-induced cultures, by evaluating three glycerol feeding profiles-constant, exponential, and gradient-during the pre-induction phase. Interestingly, different glycerol feeding profiles produced differently processed STEAP1. This platform was optimized using a combination of chemical chaperones for ensuring the structural stabilization and appropriate processing of the target protein. The supplementation of culture medium with 6 % (v/v) DMSO and 1 M proline onto a gradient glycerol/constant methanol feeding promoted increased biosynthesis levels of STEAP1 and minimized aggregation events. Deglycosylation assays with peptide N-glycosidase F showed that glycerol constant feed is associated with an N-glycosylated pattern of STEAP1. The biological activity of recombinant STEAP1 was also validated, once the protein enhanced the proliferation of LNCaP and PC3 cancer cells, in comparison with non-tumoral cell cultures. This methodology could be a crucial starting point for large-scale production of active and stable conformation of recombinant human STEAP1. Thus, it could open up new strategies to unveil the structural rearrangement of STEAP1 and to better understand the biological role of the protein in cancer onset and progression.

The present work shows the application of nickel- and magnesium-crosslinked gellan microspheres in ionic and affinity capture strategies to directly extract hSCOMT from the complex Komagataella pastoris lysate through a simple batch method. Both formulations present similar morphology, but nickel–crosslinked microspheres present higher crosslinker content and smaller diameters. Four different capture strategies were established, by manipulating the ionic strength, pH, temperature and competing agents' presence. The most promising results for hSCOMT capture and clarification were obtained employing an ionic strategy with nickel-crosslinked microspheres and an affinity strategy with magnesium-crosslinked microspheres at 4 °C. The bioactivity results (200%) and purification degree (70%) of hSCOMT captured by the ionic strategy were more satisfactory probably due to the soft ionic conditions used (100 mM NaCl). For the first time, the gellan polysaccharide versatility was demonstrated in the microsphere application for the direct capture of hSCOMT from a complex lysate, simplifying isolation biotechnological procedures.

Therapeutic applications of plasmid DNA (pDNA) have significantly advanced during the last years. Currently, several pDNA-based drugs are already in the market, whereas several others have entered phases 2 and 3 of clinical trials. The present and future demand for pDNA requires the development of efficient bioprocesses to produce it. Commonly, pDNA is produced by cultures of Escherichia coli. It has been previously demonstrated that specific strains of E. coli with a modified substrate transport system can be able to attain high cell densities in batch mode, due to the very low overflow metabolism displayed. However, the large amounts of oxygen demanded can lead to microaerobic conditions after some hours of cultivation, even at small scale. Typically, the inherent problems for these cultures are the high oxygen demand and the accumulation of acetate, a metabolic byproduct that is synthesized aerobically when the glucose rate exceeds the limits.In recent years, several researches have been focused on the study of induction of plasmid DNA as well as strategies for fermentation using semi-defined mediums. These studies conceived relevant results that allow us to design a production platform for enhanced plasmid DNA. So, the main goal of this chapter is to show how the development of an experimental design directed to aromatic amino acids pathway can improve the yield of a therapeutic plasmid DNA by culture of a new strain of Escherichia coli VH33.

Catechol-O-methyltransferase (COMT) is an enzyme involved in catecholamine catabolism that is key for the treatment of different neurologic disorders. Actually, there are still unmet needs concerning the development of more selective membrane-bound COMT (hMBCOMT) downstream strategies, envisaging their application in structural and bio-interaction studies. Therefore, in this work, recombinant hexahistidine-tagged hMBCOMT (hMBCOMT-His6) was expressed from Pichia pastoris methanol-induced cultures in a catalytically active form (27.3 nmol h−1 mg−1 of protein) and successfully solubilized with n-dodecyl β-d-maltoside. Afterward, immobilized-metal affinity chromatography provided the required selectivity for the direct capture of hMBCOMT-His6 from detergent-solubilized P. pastoris membranes, being the target enzyme recovered in a highly purified fraction. Also, despite the relatively low purification fold (1.53), the purity of the target enzyme assessed by SDS-PAGE is high and it is recovered with biological activity (67 nmol h−1 mg−1 of protein). Then, after a final polishing stage using Q-Sepharose, a pure and immunologically active enzyme fraction was obtained. Overall, the strategy herein reported may be applied to obtain pure hMBCOMT fractions, debottlenecking the implementation of bio-interaction studies and relieving the problems associated with hMBCOMT drug discovery pipeline. In a last analysis, these studies may lead to the establishment of new pharmacological therapies, thereby improving the prognosis of neurologic disorders.

BACKGROUND Catechol-O-methyltransferase (COMT, EC 2.1.1.6) has been implicated in several human diseases including Parkinson's disease and the most appropriated therapy depends on the efficacy of the COMT inhibitors applied. Therefore, more effective drugs for COMT inhibition are important, and the development of such inhibitors requires research with recombinant COMT. RESULTS The time-course production of biologically active hexa-histidine-tagged COMT in methanol-induced Pichia pastoris cultures was evaluated and immobilized-metal affinity chromatography was applied as the main capture step for the purification of the target enzyme that proved to be extremely efficient and selective. The best strategy allowed recovering soluble COMT at 300 mmol L−1 imidazol in a highly purified fraction with a purification fold of 81 and a bioactivity recovery of 57.35%. Using this strategy, a concentration of 3.68 mg L−1 of shake-flask culture of highly purified recombinant COMT was obtained. Finally, the MALDI-TOF/TOF analysis of the purified recombinant SCOMT demonstrated that it is correctly processed since no modifications in the primary sequence were observed. CONCLUSION A new strategy was developed and implemented for the biosynthesis and purification of biologically active soluble COMT in a highly purified fraction with the ability to be inhibited by commercial Parkinson's disease inhibitors: 3,5-dinitrocatechol and entacapone. © 2016 Society of Chemical Industry

Despite technological advances, two-dimensional electrophoresis (2DE) of biological fluids, such as vitreous, remains a major challenge. In this study, artificial neural network was applied to optimize the recovery of vitreous proteins and its detection by 2DE analysis through the combination of several solubilizing agents (CHAPS, Genapol, DTT, IPG buffer), temperature, and total voltage. The highest protein recovery (94.9% ± 4.5) was achieved using 4% (w/v) CHAPS, 0.1% (v/v) Genapol, 20 mM DTT, and 2% (v/v) IPG buffer. Two iterations were required to achieve an optimized response (580 spots) using 4% (w/v) CHAPS, 0.2% (v/v) Genapol, 60 mM DTT, and 0.5% (v/v) IPG buffer at 35 kVh and 25 °C, representing a 2.4-fold improvement over the standard initial conditions of the experimental design. The analysis of depleted vitreous using the optimized protocol resulted in an additional 1.3-fold increment in protein detection over the optimal output, with an average of 761 spots detected in vitreous from different vitreoretinopathies. Our results clearly indicate the importance of combining the appropriate amount of solubilizing agents with a suitable control of the temperature and voltage to obtain high-quality gels. The high-throughput of this model provides an effective starting point for the optimization of 2DE protocols. This experimental design can be adapted to other types of matrices. Graphical abstract., CENTRO-07-ST24-FEDER-002014; CNB-CSIC is supported by grant PT13/0001, of the PE I +D+i 2013–2016, funded by ISCIII and FEDER.

Affinity chromatography strategies using amino acids as immobilize d ligands have been successfully applied for the purification of different biomolecules from complex mixtures. Therefore, in this work, several supports with immobilized amino acids were applied for the purification of membrane-bound catechol-O-methyltransferase (MBCOMT) from Pichia pastoris lysates and it was verified that l-arginine provided the required selectivity for MBCOMT isolation. The optimization of the binding and elution buffers composition allowed the recovery of purified MBCOMT in a biological and immunologically active state from the arginine support. Additional optimization experiments varying the mobile phase pH, temperature and the concentration of the injected sample were carried out and an improvement of MBCOMT adsorption and purity was observed. Indeed, the optimized conditions for MBCOMT isolation and purification consisted in: loading of 4 mg of total protein onto the column previously equilibrated at 20 °C where the target enzyme was recovered in a purified fraction using 500 mM NaCl, 10 mM DTT and 0.5 % (v/v) Triton X-100 in 10 mM Tris buffer (pH 7) with a total bioactivity recovery of 24 ± 2.2 % and a purification fold of 4.95 ± 0.23, a value that is consistent with the best values ever reported for MBCOMT. Moreover, the l-arginine support demonstrated the ability to bind the target protein in a wide range of pH values (above and below the pI of the target protein) and the MBCOMT elution occurs in a single peak pattern. Finally, the strategy here reported can aid in the implementation of crystallization studies with MBCOMT in complex with clinically relevant inhibitors since it is obtained in a purified form with biological activity. In conclusion, a novel affinity chromatography strategy was developed and implemented for recombinant MBCOMT purification in a highly immunological and biologically active state.

Therapeutic applications of plasmid DNA have significantly advanced during the last years and future demand for plasmid DNA requires the development of efficient bioprocesses. Concerning these facts, our aim is to optimize the yield of a therapeutic plasmid pcDNA3–FLAG–p53 using a new bacterial strain, Escherichia coli VH33. This innovative research combines the potential of VH33, which uses a different glucose transport allowing efficient growth and lower acetate production, for pDNA biosynthesis with a validated factorial design. This work achieved higher plasmid yields of 81.77 μg pDNA/mL, when compared with other results for similar assays which reached about 40 μg pDNA/mL. The strategy is based on the change of fermentation media composition in terms of nutrients, by the development of an experimental design directed to aromatic amino acids pathway. The results revealed which combination should be applied in terms of nitrogen and carbon source requirements in order to obtain 36.69 μg pDNA/mg cell dry mass of specific yields and 6.01% of purity on lysates. Overall, the proposal model show the influence of tyrosine, phenylalanine and tryptophan on their pathway, providing necessary precursors to nucleotides’ network, as well as temperature shift (37–42 °C) to increase the plasmid copy number per cell.

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) is an enzyme that catalyzes the methylation of catechol substrates, and while structural and functional studies of its membrane-bound isoform (MBCOMT) are still hampered by low recombinant production, Pichia pastoris has been described as an attractive host for the production of correctly folded and inserted membrane proteins. Hence, in this work, MBCOMT biosynthesis was developed using P. pastoris X33 and KM71H cells in shake flasks containing a semidefined medium with different methanol concentrations. Moreover, after P. pastoris glass beads lysis, biologically and immunologically active hMBCOMT was found mainly in the solubilized membrane fraction whose kinetic parameters were identical to its correspondent native enzyme. In addition, mixed feeds of methanol and glycerol or sorbitol were also employed, and its levels quantified using liquid chromatography coupled to refractive index detection. Overall, for the first time, two P. pastoris strains with opposite phenotypes were applied for MBCOMT biosynthesis under the control of the strongly methanol-inducible alcohol oxidase (AOX) promoter. Moreover, this eukaryotic system seems to be a promising approach to deliver MBCOMT in high quantities from fermentor cultures with a lower cost-benefit due to the cheaper cultivation media coupled with the higher titers tipically achieved in biorreactors, when compared with previously reported mammallian cell cultures., A.Q. Pedro acknowledges a doctoral fellowship (SFRH/BD/81222/2011) from Fundação para a Ciência e Tecnologia and D. Oppolzer acknowledges a fellowship (CENTRO-07-ST24_FEDER-002014- TPCR-2-004) from Programa BMais Centro^ within the scope of QREN–POPH–Advanced Formation programs cofunded by Fundo Social Europeu and MEC. This work was partially funded by Fundação para a Ciência e Tecnologia I.P. (PIDDAC) and Fundo Europeu de Desenvolvimento Regional-FEDER funds through Programa Operacional Factores de Competitividade (POFC)–COMPETE: FCOMP-01-0124-FEDER-027563 and by National Funds through FCT–Fundação para a Ciência e Tecnologia within the scope of Project BEXPL/BBB478/ BQB/0960/2012.

Nisin is a natural peptide used as a preservative in a variety of food products, in which it inhibits mainly Gram-positive bacterial growth, including multidrug-resistant pathogens. However, its application range depends on the cost-effective production and purification of this molecule. Our group has previously produced nisin by Lactococcus lactis cultivation in milk whey, which is an industrial residue from dairy production. To our knowledge, no report used milk whey as a culture medium, although several investigators have purified nisin using different techniques. We thus aimed to establish a low-cost purification of nisin obtained by this process. Samples were diluted in ammonium sulphate, applied onto \HIC\ columns (butyl sepharose \CL\ 4B matrix), and eluted with Milli-Q water or PBS. Elution fractions were monitored for protein content and nisin antibacterial activity. Water elution resulted in purification factor values (270, commercial nisin; 775, nisin produced in-house) higher than those obtained with \PBS\ elution. We concluded that purification of nisin does not require precipitation with ammonium sulphate, therefore allowing step/cost reduction. Moreover, purification from milk whey using \HIC\ provides nisin with high activity and low salt content, which can further be applied to a variety of areas., This study was supported by grants from CAPES (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior, Brazil), CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil) and FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Brazil). We thank Dr. Paulo Boschcov for his careful and critical reading of our final manuscript.

The deeper understanding of retinal detachment (RD) pathogenesis may improve the visual outcome after surgery. Given the main role of the vitreous in retinal eye diseases, two strategies were explored to identify its proteome in RD. Fractionation techniques such as anion exchange chromatography (IEX) and SDS-PAGE combined with MALDI-TOF/TOF analysis allowed to identify 127 proteins in vitreous of RD patients. From these proteins, 19 were identified using only the IEX fractionation strategy, and 117 using a bidimensional (IEX and SDS-PAGE) fractionation. Of these proteins, 68 had not yet been found in other vitreous proteomic studies. The fractionation with IEX and SDS-PAGE largely improved the number of identified proteins proving that it is crucial to combine several methodologies to cover vitreous proteome., CENTRO-07-ST24-FEDER-002014;Novartis Farma-Produtos Farmacêuticos, SA; Project POCI-01-0145-FEDER-007491

Proteomic analysis of human vitreous humor (VH) may elucidate the pathogenesis of retinal ocular diseases and may provide information for the development of potential therapeutic targets due to its pivotal location near lens and retina. The discovery of whole VH proteome involves a complex analysis of thousands of proteins simultaneously. Therefore, in proteomic studies the protein fractionation is important for reducing sample complexity, facilitating the access to the low-abundant proteins, and recognizing them as biotargets for clinical research. Although several separation methods have been used, gel-based proteomics are the most popular and versatile ones applied for global protein separation. However, chromatographic methods and its combination with other separation techniques are now beginning to be used as promising set-ups for VH protein identification. This review attempts to offer an overview of the techniques currently used with VH, exploring its methodological demands, exposing its advantages, and helping the reader to plan future experiences. Moreover, this review shows the relevance of VH proteomic analysis as a tool for the study of the mechanisms underlying some ocular diseases and for the development of new therapeutic approaches.

The development of new catechol-O-methyltransferase inhibitors has led to an improvement in the treatment of Parkinson's disease. However, despite the fact that the soluble isoform has been extensively investigated, few studies have been published concerning membrane isoform chromatographic recovery and bioactivity levels. In this work, chromatographic profiles of both catechol-O-methyltransferase isoforms were compared using quaternary amine as a ligand to evaluate its activity levels and recovery rates. Results show that both proteins required different conditions for adsorption; the soluble isoform adsorption was performed at low ionic strength, while the membrane isoform required increasing linear salt gradient. However, the application of 0.5% Triton X-100 promoted membrane isoform adsorption even at low ionic strength. Indeed, chromatographic conditions of both isoforms became similar when detergents were applied. The developed methods also appear to be highly effective in bioactivity recovery, presenting rates of 107% for soluble protein and 67 and 91% for membrane isoform without and with detergents, respectively. The chromatographic strategies with and without detergents resulted in a 4.3- and sevenfold purification, respectively, corresponding to specific activity values of 331 and 496 nmol/h/mg. Thus, the use of Q-sepharose as anion exchanger was effective in the recovery of both enzymes, which is a requirement for further kinetic and pharmacological trials.

During the last few decades, it has become evident that the compatibility of the yeast biochemical environment with the ability to process and translate the RNA transcript, along with its capacity to modify a translated protein, are relevant requirements for selecting this host cell for protein expression in several pharmaceutical and clinical applications. In particular, Pichia pastoris is used as an industrial host for recombinant protein and metabolite production, showing a powerful capacity to meet required biomolecular target production levels in high-throughput assays for functional genomics and drug screening. In addition, there is a great advantage to using P. pastoris for protein secretion, even at high molecular weights, since the recovery and purification steps are simplified owing to relatively low levels of endogenous proteins in the extracellular medium. Clearly, no single microexpression system can provide all of the desired properties for human protein production. Moreover, chemical and physical bioprocess parameters, including culture medium formulation, temperature, pH, agitation, aeration rates, induction, and feeding strategies, can highly influence product yield and quality. In order to benefit from the currently available wide range of biosynthesis strategies using P. pastoris, this mini review focuses on the developments and technological fermentation achievements, providing both a comparative and an overall integration analysis. The main aim is to highlight the relevance and versatility of the P. pastoris biosystem to the design of more cost-effective microfactories to meet the increasing demands for recombinant membrane proteins and clinical antibodies for several therapeutic applications.

The goal of this work was to develop and validate an analytical method for the detection and quantification of thebiogenicaminesserotonin(5-HT),dopamine(DA)andnorepinephrine(NE),usingmicroextractioninpacked syringe (MEPS) andliquid chromatography coupled to electrochemical detection (HPLC-ED) in urine. The method was validated according to inter-nationally accepted guidelines from the Food and Drug Administration. Linearity was established between 50 and 1000 ng/mLfor5-HTand between5and 1000ng/mLforDAand NE, withdeterminationcoefficients(R 2 )>0.99forall compounds.Thelimitsof quantification and detection were respectively 50 and 20 ng/mL for 5-HT, and 5 and 2 ng/mL for DA and NE. Within- andbetween-runprecision ranged from0.84 to 9.41%, while accuracy ranged from0.79 to 12.76%for all compounds. The interme-diate precision and accuracy were 1.50–8.36 and 0.54–13.51%, respectively. The method was found suitable for clinicalroutine analysis of the studied compounds, using a sample volume of 0.5 mL. This is thefirst study employing a commer-cially available MEPS column for the simultaneous detection and quantification of 5-HT, DA and NE in urine by coulometricdetection. Copyright © 2012 John Wiley & Sons, Ltd.Keywords: biogenic amines; urine; MEPS; HPLC-ED

In this paper, a fed-batch cultivation process in recombinant Escherichia coli BL21(DE3) bacteria, for the production of human soluble catechol-O-methyltransferase (hSCOMT), is presented. For the first time, a straightforward model is applied in a recombinant hSCOMT expression system and distinguishes an initial cell growth phase from a protein production phase upon induction. Specifically, the kinetic model predicts biomass, substrate, and product concentrations in the culture over time and was identified from a series of fed-batch experiments designed by testing several feed profiles. The main advantage of this model is that its parameters can be identified more reliably from distinct fed-batch strategies, such as glycerol pulses and exponential followed by constant substrate additions. Interestingly, with the limited amount of data available, the proposed model accomplishes satisfactorily the experimental results obtained for the three state variables, and no exhaustive process knowledge is required. The comparison of the measurement data obtained in a validation experiment with the model predictions showed the great extrapolation capability of the model presented, which could provide new complementary information for the COMT production system.

The main scope of this work is the evaluation and potential application of hydrophobic interaction chromatography in the isolation of recombinant human soluble catechol-O-methyltransferase (hSCOMT) from an Escherichia coli cell extract. Therefore, a comparative study on the interaction of recombinant hSCOMT with different hydrophobic adsorbents (butyl-, octyl-, phenyl- and epoxy-Sepharose), was developed. The four adsorbents were evaluated in terms of selectivity, recovery and fractionation of recombinant hSCOMT from its Escherichia coli-free culture broth. Our data shows that the adjustment of the ionic strength on the mobile phase and the type of hydrophobic ligand are the most useful factors for a complete binding of hSCOMT and a selective fractionation of contaminating proteins. The results of these studies demonstrate that, although epoxy-Sepharose is used as a last resort due to the high salt concentrations needed, hSCOMT bind to the other three resins at low concentrations of ammonium sulfate (≤0.6 m) and eluted just by decreasing the ionic strength on the eluent to 0 m, without loss of specific of activity. The stepwise gradient with 0.6, 0.2, 0.075 and 0 m of ammonium sulfate onto a butyl-Sepharose media was found to be the most effective in the isolation of hSCOMT, leading to an enzyme solution with a 3.9-fold increased in specific activity. Since biochemical and structural studies require significant quantities of the enzyme in an active form, the above described approach may give some insight into the optimization and development of new purification strategies of mammalian COMTs. Copyright © 2007 John Wiley & Sons, Ltd.

Membrane proteins are important drug targets in many human diseases and gathering structural information regarding these proteins encourages the pharmaceutical industry to develop new molecules using structure-based drug design studies. Specifically, membrane-bound catechol-O-methyltransferase (MBCOMT) is an integral membrane protein that catalyzes the methylation of catechol substrates and has been linked to several diseases such as Parkinson’s disease and Schizophrenia. Thereby, improvements in the clinical outcome of the therapy to these diseases may come from structure-based drug design where reaching MBCOMT samples in milligram quantities are crucial for acquiring structural information regarding this target protein. Therefore, the main aim of this work was to optimize the temperature, dimethylsulfoxide (DMSO) concentration and the methanol flow-rate for the biosynthesis of recombinant MBCOMT by Pichia pastoris bioreactor methanol-induced cultures using artificial neural networks (ANN). The optimization trials intended to evaluate MBCOMT expression by P. pastoris bioreactor cultures led to the development of a first standard strategy for MBCOMT bioreactor biosynthesis with a batch growth on glycerol until the dissolved oxygen spike, 3 h of glycerol feeding and 12 h of methanol induction. The ANN modeling of the aforementioned fermentation parameters predicted a maximum MBCOMT specific activity of 384.8 nmol/h/mg of protein at 30°C, 2.9 mL/L/H methanol constant flow-rate and with the addition of 6% (v/v) DMSO with almost 90% of healthy cells at the end of the induction phase. These results allowed an improvement of MBCOMT specific activity of 6.4-fold in comparison to that from the small-scale biosynthesis in baffled shake-flasks. The ANN model was able to describe the effects of temperature, DMSO concentration and methanol flow-rate on MBCOMT specific activity, as shown by the good fitness between predicted and observed values. This experimental procedure highlights the potential role of chemical chaperones such as DMSO in improving yields of recombinant membrane proteins with a different topology than G-coupled receptors. Finally, the proposed ANN shows that the manipulation of classic fermentation parameters coupled with the addition of specific molecules can open and reinforce new perspectives in the optimization of P. pastoris bioprocesses for membrane proteins biosynthesis.

The enzyme catechol-O-methyltransferase (COMT) plays an important role in the metabolism of catechol estrogens and degradation of the catecholamine neurotransmitters, such as epinephrine. Several analytical methods, mainly high-performance liquid chromatography with electrochemical amperometric detection, have been reported for the analysis of catecholamines and their metabolites in biological fluids. In this paper we report the relevance of controlling temperature in calibration procedures of metanephrine, an O-methylated product of catechol-O-methyltransferase, using epinephrine as substrate. The results at higher temperatures show shorter retention times of metanephrine, no undue band-broadening and increased electro signals. This study also showed that, despite different temperatures leading to similarly specific activities of recombinant human COMT as expected, there are additional advantages in flow analytical methods where good sensitivity, efficiency and selectivity is required, mainly in tissues with low levels of COMT activity.

Chitosan, a natural biopolymer presents antifungal activity that seems to be dependent on the interaction of its cationic amino groups and yeast cell surface. In this work we used ion-exchange chromatography to assess the surface charge density of Candida species and subsequently to relate this with their sensitivity profile to chitosan. The ability of several strains from distinct Candida species to interact with strong anionic and cationic exchangers was tested and the yeasts charge surface was assessed by measuring the zeta potential. Our results showed that all the yeast cells tested presented no interaction with the cationic resin and a species-related pattern of interaction was observed with the anionic resin. Specifically, regarding the Q-Sepharose support, Candida glabrata showed the lower retention affinity, followed by Candida albicans, presenting Candida tropicalis an intermediate profile; Candida parapsilosis and Candida guilliermondii revealed a stronger ionic interaction. The yeasts retention synergy in the anionic resin corroborates with the zeta potential outcomes. The behavior observed fit with sensitivity patterns to chitosan as the most susceptible species to chitosan presented higher affinity to the anionic resin in contrast to the less sensitive ones (C. albicans and C. glabrata). This data confirms and reinforces that chitosan activity is probably mediated by an ionic reaction between its amino free groups and ionic charges at the cell surface.

In the last years, chromatographic supports with amino acids as immobilized ligands (AAILs) were been used successfully for isolation of several biomolecules, such as proteins. In this context and based on specific properties of human soluble cathecol-O-methyltransferase (hSCOMT), we screened and analyzed the effect of experimental conditions, such as pH and ionic strength manipulation for hSCOMT adsorption, over six different AAIL commercial supports. Typically, the proteins adsorption on AAIL chromatographic supports is around their pI. While hSCOMT isoelectric point is around 5.5, this parameter leads us to design new adsorption strategies with several acid buffers for the chromatographic process. In terms of the ionic strength manipulation strategy, the results suggest that the AAILs–hSCOMT interaction is strongly affected by the intrinsic hSCOMT hydrophobic domains. On the other hand, the interaction mechanism of hSCOMT on amino acid resins appears to be highly dependent on the binding pH. Consequently the retention mechanism of the target enzyme on the AAILs can be as either in typical hydrophobic or ionic chromatographic supports, so long as selecting various mobile phases and separation conditions. In spite of these mixed-mode interactions and operation strategies, the elution of interferent's proteins from recombinant host can be achieved only with suitable adjusts in pH mobile phase set point. This lead to a new approach in biochromatographic COMT retention, while possess a higher specificity than other chromatographic methods reported in literature.

To develop a new stationary phase of easy production, low cost, biocompatible, biodegradable and low unspecific adsorption, a three-dimensional network was prepared by combining the natural polysaccharide of gellan with divalent cations. The stability of this cation exchange chromatographic matrix was optimized by using an experimental design tool. The optimal conditions proposed for the gellan gel formulation were 48mM ZnSO4, 0% DMF, 25°C, 0.75% gellan and 0.5h. The applicability of gellan matrix was tested by chromatographic assays with three model proteins (bovine serum albumin (BSA), α-chymotripsin and lysozyme). The results showed that the retention occurred in function of the net charge of each protein in MES buffer pH 6.2 and the elution was performed by increase of ionic strength to 750mM NaCl in MES buffer pH 6.2. Lysozyme was the more retained protein due to its positive charge more effective than α-chymotripsin, while BSA did not interact with the matrix due to its negative charge at these conditions. Dynamic binding capacity assays were accomplished to characterize this matrix and to compare with commercial resins. The values of dynamic binding capacity from gellan gel were 3.9mg/mL and 17.4mg/mL, at 10% and 50% of breakthrough, respectively. In this way, gellan gel might be a promising chromatographic matrix to explore ionic interactions and to be applied in different purification strategies, getting the best benefit from its use at low cost.

Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. While large amounts of purified proteins are required for pharmaceutical and crystallization attempts, there is an unmet need for the development of novel heterologous membrane protein overexpression systems. Specifically, we tested the application of Brevibacillus choshinensis cells for the biosynthesis of human membrane bound catechol-O-methyltransferase (hMBCOMT). In terms of the upstream stage moderate to high expression was obtained for complex media formulation with a value near 45 nmol/h/mg for hMBCOMT specific activity achieved at 20 h culture with 37°C and 250 rpm. Subsequently, the efficiency for reconstitution of hMBCOMT is markedly null in the presence of ionic detergents, such as sodium dodecyl sulphate (SDS). In general, for non-ionic and zwiterionic detergents, until a detergent critic micellar concentration (CMC) of 1.0 mM, hMBCOMT shows more biological activity at lower detergent concentrations while for detergent CMC higher than 1 mM, higher detergent concentrations seem to be ideal for hMBCOMT solubilization. Indeed, from the detergents tested, the non-ionic digitonin at 0.5% (w/v) appears to be the most suitable for hMBCOMT solubilization.

Sodium citrate (SC) and low temperatures between 7 and 5 degrees C are effective in suppressing aggregation of proteins and may be beneficial to be included during a purification process. In this work, we analyzed the application of dual salt system, ammonium sulfate (AS) and SC on binding and elution conditions of recombinant hSCOMT on typical HIC sorbents. Specifically in butyl and octyl supports, the use of, respectively, 300 mM AS/200 mM SC and 25 mM AS/25 mM SC in the loading buffer resulted in complete binding of COMT. Elution was obtained by decreasing the ionic strength to 0 M of salt. For the delineate goal, it also favorably increased the support chain length while a consequent decrease in the dual ionic strength was observed for hSCOMT retention. In the presence of dual salt systems octyl media exhibited classic HIC behavior, good protein selectivity, an excellent purification factor and reduced denaturation effects of hSCOMT observed with higher salt concentrations. Also the inclusion of temperature control during the elution step appears to be advantageous for greater activity recovery without enzyme aggregation. In fact, these results could allow the prediction of most stabilizing conditions for this termolabile enzyme on the chromatographic stage, regarding salt types and therefore effectiveness to improve HIC selectivity and desirable purity on the target fractions.

The obtention of high yields of purified plasmid DNA is viewed as an essential issue to be considered towards efficient production of DNA vaccines and therapeutic plasmids. In this work, Escherichia coli DH5alpha bearing the pVAX1-LacZ plasmid was grown in a developed semi-defined medium at different temperatures and tryptone concentrations. Analysis of pDNA yields and E. coli morphology revealed that at higher temperatures (37 and 40 degrees C), higher specific yields and E. coli filamentation were obtained. However, the best results were achieved when a lower tryptone concentration was used. This approach was shown to be a powerful tool to promote plasmid amplification, keeping the desirable plasmid structure, and favoring the attainment of quality. Our results suggest that by using tryptone alone as an amino acid source, pDNA amplification was improved and a specific yield of 20.43 mg pDNA/g dcw was achieved, proving that this strategy can improve pDNA yield even at a small scale.

A panel of four hydrophobic adsorbents (butyl-, octyl-, phenyl- and epoxy-Sepharose) was used to examine the selectivity and fractionation of several proteose peptone 3 (PP3) forms from a freeze-dried extract of whey bovine milk. In parti- cular, the effects of altering the ligand type and salt were investigated. The chromatographic studies suggest that PP3 strongly interacts among the three commercial hydrophobic resins leading to a drop off in selectivity, while a complete binding was achieved at low salt concentrations (below 0.5 M) and total elution only with phosphate buffer and/or water stepwise conditions. Only in epoxy-Sepharose was an appreciably selectivity of the several fractions of PP3 present in the initial feedstock attained. Despite the high salt concentration for a complete binding of PP3 (above 1.5 M ammonium sulfate) onto this support, the dual salt system (ammonium sulfate 1 M and sodium citrate 0.8 M) led to a high separation degree of high and low molecular weight forms of PP3. Copyright © 2008 John Wiley & Sons, Ltd.

Catechol- O -methyltransferase (COMT) is a significant target in protein engineering due to its role not only in normal brain function but also to its possible involvement in some human disorders. In this work, a new approach was employed for the purification of recombinant human soluble COMT (hSCOMT) using hydrophobic interaction chromatography, as the main isolation method, from an Escherichia coli culture broth. A simplified overall process flow is proposed. Indeed, with an optimized heterologous expression system for recombinant hSCOMT production, such as E. coli , it was possible to produce and recover the active monomeric enzyme directly from the cell crude culture broth either by a freeze/thaw or ultrasonication lysis step. The recombinant enzyme present in the bacterial soluble fraction, exhibited similar affinity for epinephrine ( K m 276 [215; 337] μM) and the methyl donor ( S -adenosyl- l -methionine, SAMe) ( K m 36 [30; 41] μM) as human SCOMT. After the precipitation step by 55% of ammonium sulphate, a HIC step on the butyl-sepharose resin was found to be highly effective in selectively eluting a range of contaminating key proteins present in the concentrate soluble extract. Consequently, the partially purified eluate from HIC could then be loaded and polished by gel filtration in order to increase the process efficiency. The final product appeared as a single band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The procedure resulted in a global 10.9-fold purification with a specific activity of 5500 nmol/h/mg of protein. The widespread applicability of the process, here described, to different COMT sources could make this protocol highly useful for all studies requiring purified and active COMT proteins.

Highlights • Constant feeds perform better than exponential feeds for hSCOMT production. • A constant feed of 1 g/L/h yielded 40 OD600 and a hSCOMT activity of 442 nmol/h/mg. • A high percentage of viability was maintained in constant fed-batch fermentations., Catechol-O-methyltransferase (COMT, EC 2.1.1.6) plays a crucial role in dopamine metabolism which has intimately linked this enzyme to some neurodegenerative diseases, such as Parkinson's disease. In recent years, in the attempt of developing new therapeutic strategies for Parkinson's disease, there has been a growing interest in the search for effective COMT inhibitors. In order to do so, large amounts of COMT in an active form are needed, and the best way to achieve this is by up-scaling its production through biotechnological processes. In this work, a fed-batch process for the biosynthesis of the soluble isoform of COMT in Escherichia coli is proposed. This final process was selected through the evaluation of the effect of different dissolved oxygen concentrations, carbon and nitrogen source concentrations and feeding profiles on enzymatic production and cell viability, while controlling various parameters (pH, temperature, starting time of the feeding and induction phases and carbon source concentration) during the process. After several batch and fed-batch experiments, a final specific COMT activity of 442.34 nmol/h/mg with approximately 80% of viable cells at the end of the fermentation were achieved. Overall, the results described herein provide a great improvement on hSCOMT production in recombinant bacteria and provide a new and viable option for the use of a fed-batch fermentation with a constant feeding profile to the large scale production of this enzyme.