RESUMO
Our objective was to investigate the Escherichia coli localization (such as supernatant, cytoplasm and inclusion bodies) of an anti-alphaIIb-beta3 (alphaIIbbeta3) scFv fragment referred to as scFv[EBB3] produced in batch fermentation. Immobilized metal affinity chromatography (IMAC) purification was performed on supernatant using expanded bed absorbed technology (EBA) and on sonicated cells in native conditions over an immobilized copper-ion affinity column. Inclusion bodies were solubilized before IMAC purification and the refolding procedure was performed on the column. The majority of scFv[EBB3] were present as inclusion bodies (55%), whereas 36% were found in the cytoplasm and only 9% secreted in the supernatant. The scFv activity was assessed by enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunohistochemistry analyses performed on a thrombus induced in vivo on an atherosclerotic rabbit model.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Escherichia coli/crescimento & desenvolvimento , Região Variável de Imunoglobulina/isolamento & purificação , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Aterosclerose/imunologia , Cromatografia Líquida , Modelos Animais de Doenças , Escherichia coli/química , Humanos , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Corpos de Inclusão/química , Corpos de Inclusão/genética , Corpos de Inclusão/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Dobramento de Proteína , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Trombose/induzido quimicamente , Trombose/imunologiaRESUMO
The enhanced green fluorescent protein (EGFP) was over-expressed in Escherichia coli as inclusion bodies to increase its quantity and to facilitate its purification. Insoluble EGFP has been purified on Q Hyper Z matrix by expanded bed adsorption after solubilization in 8 M urea. The adsorption was made in expanded bed mode to avoid centrifugation. EBA-column refolding was done by elimination of urea and elution with NaCl. The EGFP was obtained as a highly purified soluble form with similar behavior in fluorescence and electrophoresis as native EGFP.
Assuntos
Cromatografia por Troca Iônica/métodos , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/isolamento & purificação , Dobramento de Proteína , Adsorção , Eletroforese em Gel de Poliacrilamida , Escherichia coli/ultraestrutura , Proteínas de Fluorescência Verde/biossíntese , Corpos de Inclusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Solubilidade , Espectrometria de Fluorescência , UreiaRESUMO
Production of anti-alphaIIbbeta3 (anti-alphaIIbbeta3)-binding single-chain FV (scFv) fragments obtained from combinatorial libraries of IgG human antibodies is of broad interest for imaging and treatment of acute coronary syndromes. The objective of our work was to design an optimized production of one selected anti-alphaIIbbeta3-binding scFv fragment for subsequent in vivo animal studies. Fed-batch fermentation was initiated with 2TY media supplemented with 0.1 M glucose. This growing batch culture was used as a starting point for further fed-batch induction, in which a media without glucose containing 1 mM IPTG and 0.4 M saccharose was continuously added. Subsequent purification was performed on the whole cell extract in native conditions over an immobilized copper-ion affinity column. The improved conditions allowed the recovery of 5 mg of highly purified scFv fragments as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bioactivity of the scFv fragments was further monitored by ELISA, cytometric and immunohistochemical methods.
Assuntos
Cromatografia de Afinidade/métodos , Fragmentos de Imunoglobulinas/isolamento & purificação , Região Variável de Imunoglobulina/isolamento & purificação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Animais , Plaquetas/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Fermentação , Citometria de Fluxo , Imuno-Histoquímica , Masculino , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/farmacologia , Coelhos , Trombose/imunologia , TransfecçãoRESUMO
Three anion exchanger expanded bed adsorption (EBA) matrices: Streamline DEAE, Streamline Q XL and Q Hyper Z were evaluated with the aid of EFGP from an ultrasonic homogenate of Escherichia coli. Two pH of buffer were tested. Capture was done in an expanded mode whereas elution was done in a packed mode. The same conditions were chosen for evaluation of the three matrices. We observed a loss of EGFP (8-15%) in the through flow fraction especially with the Streamline Q XL matrix, probably due to an aggregation of beads during sample application. The beads of this matrix possess tentacles which probably retain a lot of cellular and molecular debris. The two other matrices gave a good purification of the EGFP (7-15-fold) but the Q Hyper Z matrix appeared to give the best results. It is composed of little size and density beads which lead to a higher exchange surface and then a better mass transfer.
Assuntos
Resinas de Troca Aniônica , Cromatografia por Troca Iônica/instrumentação , Proteínas de Fluorescência Verde/química , Adsorção , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
The aim of this work was to test a chromatographic support, 4-mercaptoethyl pyridine (4-MEP) Hypercel, for penicillin acylase purification by using pure penicillin acylase and crude extract. Two equilibration buffers with various salt concentrations and different flow rates were tested. The relationships between electrostatic and hydrophobic interactions and proteins are demonstrated. (NH4)2SO4 proved preferable because no salting-in occurred, contrary to NaCl. The recovery and purification fold were similar to those obtained in pseudo-affinity chromatography with a three-fold reduction of the (NH4)2SO4 concentration.
Assuntos
Cromatografia Líquida/métodos , Penicilina Amidase/isolamento & purificação , Soluções Tampão , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Penicilina Amidase/metabolismo , Piridinas/químicaRESUMO
The aim of this work was to test a recycling method for imidazole used in immobilized metal affinity chromatography (IMAC) as eluent for recombinant histidine-tag (His-tag) protein. After evaluating two supports, the method was optimized with a mixture of bovine serum albumin, sodium chloride and imidazole. Recycling was performed with an eluate fraction from IMAC of His-tag enhanced green fluorescent protein produced in our laboratory and pure imidazole was recovered in water and was analyzed after being freeze-dried. The imidazole was then reused as eluent in IMAC without any modification in its structure or behavior. This procedure can be used for large-scale chromatography.
Assuntos
Cromatografia de Afinidade/métodos , Histidina/química , Imidazóis/análise , Proteínas Recombinantes/isolamento & purificação , Reprodutibilidade dos TestesRESUMO
In this report, we describe a two-step chromatographic procedure for the purification of His-tag EGFP by immobilized metal affinity expanded bed adsorption (IMAEBA) as the capture step and size exclusion chromatography as the polishing step. The use of proteins including a histidine-tag facilitates their subsequent purification after expression in many microorganisms. This meets the needs of scientific researchers as well as industrialists in purifying recombinant proteins. The procedure described allowed the obtention of 230 mg pure EGFP from 1 l simple batch culture with a recovery of 90%.
Assuntos
Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Cromatografia de Afinidade , Cromatografia em Gel , Clonagem Molecular , DNA , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Histidina/química , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/químicaRESUMO
In this report, we describe a new process for the on-line purification of His-tag EGFP (enhanced green fluorescent protein) taken directly from a bioreactor by continuous ultrasonic homogenization coupled with immobilized metal affinity expanded bed adsorption (IMAEBA). The use of proteins including a histidine-tag facilitates their subsequent purification after expression in many microorganisms. This meets the needs of scientific researchers as well as industrialists interested in purifying recombinant proteins. After evaluating the different flow-rates and ultrasonic probe sizes, the on-line purification was tested. After ultrasonic treatment, 70% of the cells were broken and 90% of free EGFP was recovered after IMAEBA. In our conditions, more than 450 mg of EGFP were obtained in 15 h. On-line bioreactor-ultrasonic probe-immobilized metal affinity expanded bed adsorption is a rapid automated technique for obtaining large quantities of pure EGFP.
Assuntos
Reatores Biológicos , Cromatografia de Afinidade/métodos , Histidina/química , Proteínas Luminescentes/isolamento & purificação , Adsorção , Eletroforese em Gel de Poliacrilamida , Proteínas de Fluorescência Verde , Proteínas Luminescentes/química , Metais/química , UltrassomRESUMO
The activity of the plant plasma membrane (PM) H(+)-ATPase was studied with fresh, cut or aged tissues of sugar beet (Beta vulgaris L.) leaves. The rate of acidification of the medium by tissue samples was strongly stimulated by ageing, but unaffected by cutting. The proton-pumping activity and the specific activity of the vanadate-sensitive ATPase of purified PM vesicles prepared from aged tissues were much higher than that of fresh tissues, whereas cutting had no effect. Yet, both ageing and cutting increased the amount of PM H(+)-ATPase detected by enzyme-linked immunosorbent assays. Likewise, both ageing and cutting increased the levels of pma4 and pma2 ATPase transcripts, as assayed with the corresponding probes from Nicotiana plumbaginifolia. Ageing increases, within a few hours, the levels of the transcripts, the translation and the activity of several PM H(+)-ATPase families. Cutting, which represents a milder mechanical stress, only increases the levels of the transcripts and their translation, without detectable effect on the activity at the biochemical or physiological level, which suggests a post-translational control of this activity. Thus, upon mechanical stress, the activity of the H(+)-ATPase, a key enzyme of the plant PM is rapidly and tightly regulated by transcriptional and post-translational controls.
Assuntos
Membrana Celular/enzimologia , Regulação da Expressão Gênica de Plantas , Plantas/enzimologia , Biossíntese de Proteínas , ATPases Translocadoras de Prótons/genética , Transcrição Gênica , Concentração de Íons de Hidrogênio , Cinética , Estimulação Física , Folhas de Planta/enzimologia , Plantas/genética , Plantas/ultraestrutura , Plantas Tóxicas , Fatores de Tempo , Nicotiana , Vanadatos/farmacologiaRESUMO
Purified plasma membranes from sugar beet leaves were solubilized by 1% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate and loaded on a sepharose 6 B column substituted with sucrose. Elution with sucrose at pH 5.2 yielded a peak that represented 0.2% of the loaded protein. This peak did not appear when the samples were pretreated with either 0.5 mM N-ethylmaleimide (NEM) or 0.5 mM para-chloromercuribenzenesulfonic acid. It was also absent when palatinose, a sucrose analogue not recognized by the sucrose transporter, was used as the affinity ligand. The peak specifically eluted by sucrose from the sucrose-Sepharose column exhibited sucrose transport activity after reconstitution into proteoliposomes. This peak was further fractionated by ion-exchange chromatography on a Mono-Q column, and the different fractions obtained were differentially labeled by [3H]NEM in the presence of sugars recognized (sucrose, maltose) or not recognized (palatinose) by the sucrose transporter. The data allowed to identify two fractions that were enriched with two polypeptides (56 and 41 kDa) differentially labeled by NEM in the presence of sucrose.