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1.
Methods Mol Biol ; 2178: 285-299, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33128756

RESUMO

In downstream processing, large-scale chromatography plays an important role. For its development, screening experiments followed by pilot-plant chromatography are mandatory steps. Here we describe fast, simple, and inexpensive methods for establishing a preparative chromatography for the separation of complex protein mixtures, based on sample displacement batch chromatography. The methods are demonstrated by anion-exchange chromatography of a human plasma protein fraction (Cohn IV-4), including the screening step and upscaling of the chromatography by a factor of one hundred. The results of the screening experiments and the preparative chromatography are monitored by SDS-PAGE electrophoresis. In summary, we provide a protocol, which should be easily adaptable for the chromatographic large-scale purification of other proteins, in the laboratory as well as in the manufacturing of biopharmaceuticals. These protocols cover the initial piloting steps for establishing a large-scale sample batch chromatography. The results from the piloting steps may also be applied for packed columns for performing simulated-moving-bed (SMB) chromatography rather than batch chromatography.


Assuntos
Albumina Sérica Humana/química , Albumina Sérica Humana/isolamento & purificação , Soroglobulinas/química , Soroglobulinas/isolamento & purificação , Cromatografia por Troca Iônica , Humanos
2.
Mol Microbiol ; 103(5): 860-874, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27997732

RESUMO

The otherwise harmless skin inhabitant Staphylococcus epidermidis is a major cause of healthcare-associated medical device infections. The species' selective pathogenic potential depends on its production of surface adherent biofilms. The Cell wall-anchored protein Aap promotes biofilm formation in S. epidermidis, independently from the polysaccharide intercellular adhesin PIA. Aap requires proteolytic cleavage to act as an intercellular adhesin. Whether and which staphylococcal proteases account for Aap processing is yet unknown. Here, evidence is provided that in PIA-negative S. epidermidis 1457Δica, the metalloprotease SepA is required for Aap-dependent S. epidermidis biofilm formation in static and dynamic biofilm models. qRT-PCR and protease activity assays demonstrated that under standard growth conditions, sepA is repressed by the global regulator SarA. Inactivation of sarA increased SepA production, and in turn augmented biofilm formation. Genetic and biochemical analyses demonstrated that SepA-related induction of biofilm accumulation resulted from enhanced Aap processing. Studies using recombinant proteins demonstrated that SepA is able to cleave the A domain of Aap at residue 335 and between the A and B domains at residue 601. This study identifies the mechanism behind Aap-mediated biofilm maturation, and also demonstrates a novel role for a secreted staphylococcal protease as a requirement for the development of a biofilm.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Metaloendopeptidases/metabolismo , Processamento de Proteína Pós-Traducional , Staphylococcus epidermidis/enzimologia , Staphylococcus epidermidis/fisiologia , Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Polissacarídeos Bacterianos/metabolismo , Ligação Proteica , Staphylococcus epidermidis/química , Staphylococcus epidermidis/genética
3.
Methods Mol Biol ; 1129: 325-38, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24648085

RESUMO

In downstream processing large scale chromatography plays an important role. For its development screening experiments followed by pilot plant chromatography are mandatory steps. Here we describe fast, simple, and inexpensive methods for establishing a preparative chromatography for the separation of complex protein mixtures, based on sample displacement batch chromatography. The methods are demonstrated by anion-exchange chromatography of a human plasma protein fraction (Cohn IV-4), including the screening step and scaling up of the chromatography by a factor of 100. The results of the screening experiments and the preparative chromatography are monitored by SDS-PAGE electrophoresis. In summary we provide a protocol which should be easily adaptable for the chromatographic large scale purification of other proteins, in the laboratory as well as in industry for commercial manufacturing. For the latter these protocols cover the initial piloting steps for establishing a sample batch chromatography based on packed columns rather than batch chromatography.


Assuntos
Proteínas Sanguíneas/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Eletroforese em Gel de Poliacrilamida , Humanos
4.
Mol Microbiol ; 86(2): 394-410, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22957858

RESUMO

Biofilm formation is essential for Staphylococcus epidermidis pathogenicity in implant-associated infections. Nonetheless, large proportions of invasive Staphylococcus epidermidis isolates fail to form a biofilm in vitro. We here tested the hypothesis that this apparent paradox is related to the existence of superimposed regulatory systems suppressing a multicellular biofilm life style in vitro. Transposon mutagenesis of clinical significant but biofilm-negative S. epidermidis 1585 was used to isolate a biofilm positive mutant carrying a Tn917 insertion in sarA, chief regulator of staphylococcal virulence. Genetic analysis revealed that inactivation of sarA induced biofilm formation via overexpression of the giant 1 MDa extracellular matrix binding protein (Embp), serving as an intercellular adhesin. In addition to Embp, increased extracellular DNA (eDNA) release significantly contributed to biofilm formation in mutant 1585ΔsarA. Increased eDNA amounts indirectly resulted from upregulation of metalloprotease SepA, leading to boosted processing of autolysin AtlE, in turn inducing augmented autolysis and release of eDNA. Hence, this study identifies sarA as a negative regulator of Embp- and eDNA-dependent biofilm formation. Given the importance of SarA as a positive regulator of polysaccharide mediated cell aggregation, the regulator enables S. epidermidis to switch between mechanisms of biofilm formation, ensuring S. epidermidis adaptation to hostile environments.


Assuntos
Adesinas Bacterianas/genética , Proteínas de Bactérias/metabolismo , Bacteriólise , Biofilmes , DNA Bacteriano/metabolismo , Regulação para Baixo , Regulação Bacteriana da Expressão Gênica , Staphylococcus epidermidis/fisiologia , Transativadores/metabolismo , Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Humanos , Staphylococcus epidermidis/genética , Transativadores/genética
5.
J Sep Sci ; 35(22): 3170-6, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22707445

RESUMO

Displacement chromatography has been shown to be an effective alternative for protein purification. We investigated in this study sample displacement chromatography, which does not require a displacer molecule. Furthermore, we performed a screening for determination of parameters for an optimal sample displacement chromatography. We screened the affinities of cytochrome C, lysozyme, myoglobin, and ribonuclease A toward a cation exchange material as a function of different pH values and to presence of different concentrations of sodium chloride in the sample application buffer. Sample displacement chromatography in batch chromatography mode for the separation of the protein mixture was studied with a sample application buffer with a pH of 5 and 7. As predicted by the screening experiments, sample displacement chromatography was most effective at pH 7 since this pH guaranteed the largest differences of the affinities of the four proteins toward the stationary phase. In summary, we describe here sample displacement chromatography in the batch chromatography mode for the separation of proteins, which is a simple and fast alternative to conventional displacement chromatography. Systematic screening of chromatographic parameters prior to sample displacement chromatography promises a successful separation of a target protein.


Assuntos
Resinas de Troca de Cátion/química , Cromatografia por Troca Iônica/métodos , Proteínas/isolamento & purificação , Adsorção , Cromatografia por Troca Iônica/instrumentação , Concentração de Íons de Hidrogênio , Proteínas/química
6.
Artigo em Inglês | MEDLINE | ID: mdl-22727752

RESUMO

Liquid chromatography is often the method of choice for the analysis of proteins in their native state. Nevertheless compared to two-dimensional electrophoresis, the resolution of common chromatographic techniques is low. Liquid chromatography in the displacement mode has previously been shown to offer higher resolution and to elute proteins in the high concentrations. In this study we compared to what extend displacement mode was a suitable alternative to gradient mode for the separation of a complex protein mixture using anion-exchange displacement chromatography and if it is therefore helpful for proteomic investigations. Hence we analyzed the qualitative protein composition of each fraction by tryptic digestion of the proteins, analysis of the tryptic peptides by liquid chromatography coupled to mass spectrometry followed by data base analysis and by measuring the elution profiles of 22 selected proteins with selected reaction monitoring mass spectrometry. In the fractions of displacement mode a significantly higher number of identified proteins (51 versus 16) was yielded in comparison to gradient mode. The resolution of displacement chromatography was slightly lower than of gradient chromatography for many but not for all proteins. The selectivities of displacement mode and gradient mode are very different. In conclusion displacement chromatography is a well suited alternative for top-down proteomic approaches which start with separating intact proteins first prior to mass spectrometric analysis of intact or digested proteins. The significant orthogonality of both modes may be used in the future for combining them in multidimensional fractionation procedures.


Assuntos
Proteínas Sanguíneas/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Espectrometria de Massas/métodos , Ânions/química , Proteínas Sanguíneas/química , Condutividade Elétrica , Humanos , Espectrofotometria Ultravioleta
7.
Int J Mol Sci ; 11(9): 3122-37, 2010 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-20957083

RESUMO

A new assay system for chitin has been developed. It comprises the chitin-binding protein ChbB in fusion with a His-tag as well as with a Strep-tag, the latter of which was chemically coupled to horseradish peroxidase. With the resulting complex, minimal quantities of chitin are photometrically detectable. In addition, the assay allows rapid scoring of the activity of chitin-synthases. As a result, a refined procedure for the rapid purification of yeast chitosomes (nano-machineries for chitin biosynthesis) has been established. Immuno-electronmicroscopical studies of purified chitosomes, gained from a yeast strain carrying a chitin-synthase gene fused to that for GFP (green-fluorescence protein), has led to the in situ localization of chitin-synthase-GFP molecules within chitosomes.


Assuntos
Quitina Sintase/análise , Quitina/análise , Proteínas de Saccharomyces cerevisiae/análise , Saccharomyces cerevisiae/química , Imunoensaio/métodos , Saccharomyces cerevisiae/ultraestrutura
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