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1.
Protein Expr Purif ; 94: 67-72, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24215862

RESUMO

The lactose autoinduction system for recombinant protein production was combined with enzymatic glucose release as a method to provide a constant feed of glucose instead of using glycerol as a carbon substrate. Bioreactor cultivation confirmed that the slow glucose feed does not prevent the induction by lactose. HPLC studies showed that with successful recombinant protein production only a very low amount of lactose was metabolized during glucose-limited fed-batch conditions by the Escherichia coli strain BL21(DE3)pLysS in well-aerated conditions, which are problematic for glycerol-based autoinduction systems. We propose that slow enzymatic glucose feed does not cause a full activation of the lactose operon. However recombinant PDI-A protein (A-domain of human disulfide isomerase) was steadily produced until the end of the cultivation. The results of the cultivations confirmed our earlier observations with shaken cultures showing that lactose autoinduction cultures based on enzymatic glucose feed have good scalability, and that this system can be applied also to bioreactor cultivations.


Assuntos
Reatores Biológicos , Glucose/metabolismo , Lactose/metabolismo , Proteínas Recombinantes/biossíntese , Meios de Cultura/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glucose/farmacologia , Humanos , Lactose/farmacologia , Pró-Colágeno-Prolina Dioxigenase/biossíntese , Pró-Colágeno-Prolina Dioxigenase/genética , Isomerases de Dissulfetos de Proteínas/biossíntese , Isomerases de Dissulfetos de Proteínas/genética , Proteínas Recombinantes/genética
2.
Bioprocess Biosyst Eng ; 37(7): 1261-9, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24326738

RESUMO

Recombinant protein synthesis in Pichia pastoris is generally controlled by the strong methanol inducible AOX1 promoter which is repressed by glucose and glycerol. In shake flasks, commonly one or two methanol pulses are added per day for induction. Such pulse feeding procedure leads to carbon starvation phases, which may enhance proteolytic activities and, therefore, cause product losses. Starvation between the methanol pulses could be avoided with a continuous enzymatic feed of glucose from a glucose-based polymer. The amount of glucose was low enough to prevent AOX1 repression by glucose. Energy and carbon were continuously supplied for cell maintenance resulting in significantly increased cell densities and product activities, as shown here at the example of a fungal lipase expressed in P. pastoris. A threefold improvement in measured product activity was obtained by applying enzymatic glucose feed and a further improvement was achieved by applying a defined mixture of ammonium compounds. The strategy described here simplifies the general procedure in shaken cultures by allowing the direct continuation of the cultivation from glucose to the methanol-based production phase without a medium change. It is easily applicable to multiwell plates and thus beneficial for high throughput applications.


Assuntos
Oxirredutases do Álcool/química , Proteínas Fúngicas/química , Glucose/química , Pichia/metabolismo , Ácidos/química , Compostos de Amônio/química , Biotecnologia/métodos , Concentração de Íons de Hidrogênio , Lipase/metabolismo , Metanol/química , Polímeros/química , Fatores de Tempo
3.
Protein Expr Purif ; 91(2): 147-54, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23938950

RESUMO

Recombinant protein expression from lac derived promoters by the autoinduction regime is based on diauxic growth of Escherichia coli on glucose and lactose. Glycerol is used as a supporting carbon source during the lactose-induced expression. While this glycerol-based formulation usually provides high cell densities, successful protein expression by autoinduction is often very dependent on correct aeration level. This complicates the reproducibility and scalability of the cultures. In this study we investigate the use of an alternative autoinduction formulation, in which the supporting carbon source is provided by fed-batch-like slow glucose feed from a biocatalytically degraded polysaccharide. The glucose feed as supporting carbon source allowed for high level of autoinduced target protein expression from T7lac promoter in E. coli BL21(DE3) and from T5lac promoter in E. coli K-12 RB791(lacI(q)) with lactose concentrations of 0.5-2gl(-1). Cell densities and protein yields per culture volume were similar to or higher than in the glycerol-based ZYM-5052 medium. In the glycerol-based medium, protein production was adversely influenced by high aeration level, resulting in 75-90% reduction in protein yield per cell compared to more moderately aerated conditions. The glucose fed-batch medium attenuated this oxygen-sensitivity and provided robust high-yield expression also under high aeration rates. It is concluded that the slow glucose feed as supporting carbon source mitigates aeration-related scale differences in autoinduced protein expression, and combined with the benefit of high product yields this makes the fed-batch autoinduction medium ideal for high-throughput screening and scale-up of the production process.


Assuntos
Glucose/metabolismo , Lactose/metabolismo , Oxigênio/metabolismo , Proteínas Recombinantes/biossíntese , Aerobiose , Carbono/metabolismo , Meios de Cultura , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
4.
Microb Cell Fact ; 12: 73, 2013 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-23895637

RESUMO

BACKGROUND: Fab antibody fragments in E. coli are usually directed to the oxidizing periplasmic space for correct folding. From periplasm Fab fragments may further leak into extracellular medium. Information on the cultivation parameters affecting this leakage is scarce, and the unpredictable nature of Fab leakage is problematic regarding consistent product recovery. To elucidate the effects of cultivation conditions, we investigated Fab expression and accumulation into either periplasm or medium in E. coli K-12 and E. coli BL21 when grown in different types of media and under different aeration conditions. RESULTS: Small-scale Fab expression demonstrated significant differences in yield and ratio of periplasmic to extracellular Fab between different culture media and host strains. Expression in a medium with fed-batch-like glucose feeding provided highest total and extracellular yields in both strains. Unexpectedly, cultivation in baffled shake flasks at 150 rpm shaking speed resulted in higher yield and accumulation of Fabs into culture medium as compared to cultivation at 250 rpm. In the fed-batch medium, extracellular fraction in E. coli K-12 increased from 2-17% of total Fab at 250 rpm up to 75% at 150 rpm. This was partly due to increased lysis, but also leakage from intact cells increased at the lower shaking speed. Total Fab yield in E. coli BL21 in glycerol-based autoinduction medium was 5 to 9-fold higher at the lower shaking speed, and the extracellular fraction increased from ≤ 10% to 20-90%. The effect of aeration on Fab localization was reproduced in multiwell plate by variation of culture volume. CONCLUSIONS: Yield and leakage of Fab fragments are dependent on expression strain, culture medium, aeration rate, and the combination of these parameters. Maximum productivity in fed-batch-like conditions and in autoinduction medium is achieved under sufficiently oxygen-limited conditions, and lower aeration also promotes increased Fab accumulation into extracellular medium. These findings have practical implications for screening applications and small-scale Fab production, and highlight the importance of maintaining consistent aeration conditions during scale-up to avoid changes in product yield and localization. On the other hand, the dependency of Fab leakage on cultivation conditions provides a practical way to manipulate Fab localization.


Assuntos
Escherichia coli/metabolismo , Fragmentos Fab das Imunoglobulinas/metabolismo , Oxigênio/metabolismo , Técnicas de Cultura Celular por Lotes , Meios de Cultura/farmacologia , Escherichia coli/crescimento & desenvolvimento , Concentração de Íons de Hidrogênio , Fragmentos Fab das Imunoglobulinas/genética , Periplasma/efeitos dos fármacos , Periplasma/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
5.
Microb Cell Fact ; 10: 107, 2011 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-22152005

RESUMO

This report describes the combined use of an enzyme-based glucose release system (EnBase®) and high-aeration shake flask (Ultra Yield Flask™). The benefit of this combination is demonstrated by over 100-fold improvement in the active yield of recombinant alcohol dehydrogenase expressed in E. coli. Compared to Terrific Broth and ZYM-5052 autoinduction medium, the EnBase system improved yield mainly through increased productivity per cell. Four-fold increase in oxygen transfer by the Ultra Yield Flask contributed to higher cell density with EnBase but not with the other tested media, and consequently the product yield per ml of EnBase culture was further improved.


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Escherichia coli/metabolismo , Glucose/metabolismo , Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cultura Celular por Lotes/instrumentação , Meios de Cultura/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Fermentação , Lactobacillus/enzimologia , Proteínas Recombinantes/genética
6.
Microb Cell Fact ; 9: 11, 2010 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-20167131

RESUMO

BACKGROUND: Cultivations for recombinant protein production in shake flasks should provide high cell densities, high protein productivity per cell and good protein quality. The methods described in laboratory handbooks often fail to reach these goals due to oxygen depletion, lack of pH control and the necessity to use low induction cell densities. In this article we describe the impact of a novel enzymatically controlled fed-batch cultivation technology on recombinant protein production in Escherichia coli in simple shaken cultures. RESULTS: The enzymatic glucose release system together with a well-balanced combination of mineral salts and complex medium additives provided high cell densities, high protein yields and a considerably improved proportion of soluble proteins in harvested cells. The cultivation method consists of three steps: 1) controlled growth by glucose-limited fed-batch to OD600 approximately 10, 2) addition of growth boosters together with an inducer providing efficient protein synthesis within a 3 to 6 hours period, and 3) a slow growth period (16 to 21 hours) during which the recombinant protein is slowly synthesized and folded. Cell densities corresponding to 10 to 15 g l(-1) cell dry weight could be achieved with the developed technique. In comparison to standard cultures in LB, Terrific Broth and mineral salt medium, we typically achieved over 10-fold higher volumetric yields of soluble recombinant proteins. CONCLUSIONS: We have demonstrated that by applying the novel EnBase Flo cultivation system in shaken cultures high cell densities can be obtained without impairing the productivity per cell. Especially the yield of soluble (correctly folded) proteins was significantly improved in comparison to commonly used LB, Terrific Broth or mineral salt media. This improvement is thought to result from a well controlled physiological state during the whole process. The higher volumetric yields enable the use of lower culture volumes and can thus significantly reduce the amount of time and effort needed for downstream processing or process optimization. We claim that the new cultivation system is widely applicable and, as it is very simple to apply, could widely replace standard shake flask approaches.


Assuntos
Técnicas de Cultura/métodos , Escherichia coli/crescimento & desenvolvimento , Biomassa , Meios de Cultura/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glucose/metabolismo , Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
7.
Artigo em Inglês | MEDLINE | ID: mdl-32760676

RESUMO

Fast, robust, and affordable antimicrobial susceptibility testing (AST) is required, as roughly 50% of antibiotic treatments are started with wrong antibiotics and without a proper diagnosis of the pathogen. Validated growth-based AST according to EUCAST or CLSI (European Committee on Antimicrobial Susceptibility Testing, Clinical Laboratory Standards Institute) recommendations is currently suggested to guide the antimicrobial therapy. Any new AST should be validated against these standard methods. Many rapid diagnostic techniques can already provide pathogen identification. Some of them can additionally detect the presence of resistance genes or resistance proteins, but usually isolated pure cultures are needed for AST. We discuss the value of the technologies applying nucleic acid amplification, whole genome sequencing, and hybridization as well as immunodiagnostic and mass spectrometry-based methods and biosensor-based AST. Additionally, we evaluate the potential of integrated systems applying microfluidics to integrate cultivation, lysis, purification, and signal reading steps. We discuss technologies and commercial products with potential for Point-of-Care Testing (POCT) and their capability to analyze polymicrobial samples without pre-purification steps. The purpose of this critical review is to present the needs and drivers for AST development, to show the benefits and limitations of AST methods, to introduce promising new POCT-compatible technologies, and to discuss AST technologies that are likely to thrive in the future.


Assuntos
Antibacterianos , Anti-Infecciosos , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana
8.
BMC Biotechnol ; 8: 33, 2008 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-18371201

RESUMO

BACKGROUND: Here we describe a new technical solution for optimization of Pichia pastoris shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in P. pastoris is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in P. pastoris is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control. RESULTS: By applying on-line pO2 monitoring we demonstrate that the widely used pulse feeding of methanol results in long phases of methanol exhaustion and consequently low expression of AOX1 controlled genes. Furthermore, we provide a solution to apply the fed-batch strategy in shake flasks. The presented solution applies a wireless feeding unit which can be flexibly positioned and allows the use of computer-controlled feeding profiles. By using the human collagen II as an example we show that a quasi-continuous feeding profile, being the simplest way of a fed-batch fermentation, results in a higher production level of human collagen II. Moreover, the product has a higher proteolytic stability compared to control cultures due to the increased expression of human collagen prolyl 4-hydroxylase as monitored by mRNA and protein levels. CONCLUSION: The recommended standard protocol for methanol addition in shake flasks using pulse feeding is non-optimal and leads to repeated long phases of methanol starvation. The problem can be solved by applying the fed-batch technology. The presented wireless feeding unit, together with an on-line monitoring system offers a flexible, simple, and low-cost solution for initial optimization of the production in shake flasks which can be performed in parallel. By this way the fed-batch strategy can be applied from the early screening steps also in laboratories which do not have access to high-cost and complicated bioreactor systems.


Assuntos
Reatores Biológicos/microbiologia , Técnicas de Cultura de Células/instrumentação , Colágeno Tipo II/metabolismo , Oxigênio/metabolismo , Pichia/enzimologia , Engenharia de Proteínas/instrumentação , Telemetria/instrumentação , Colágeno Tipo II/genética , Desenho de Equipamento , Análise de Falha de Equipamento , Análise de Injeção de Fluxo/instrumentação , Humanos , Sistemas On-Line , Oxigênio/análise , Pichia/genética
9.
Microb Cell Fact ; 7: 31, 2008 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-19017379

RESUMO

BACKGROUND: Here we describe a novel cultivation method, called EnBasetrade mark, or enzyme-based-substrate-delivery, for the growth of microorganisms in millilitre and sub-millilitre scale which yields 5 to 20 times higher cell densities compared to standard methods. The novel method can be directly applied in microwell plates and shake flasks without any requirements for additional sensors or liquid supply systems. EnBase is therefore readily applicable for many high throughput applications, such as DNA production for genome sequencing, optimisation of protein expression, production of proteins for structural genomics, bioprocess development, and screening of enzyme and metagenomic libraries. RESULTS: High cell densities with EnBase are obtained by applying the concept of glucose-limited fed-batch cultivation which is commonly used in industrial processes. The major difference of the novel method is that no external glucose feed is required, but glucose is released into the growth medium by enzymatic degradation of starch. To cope with the high levels of starch necessary for high cell density cultivation, starch is supplied to the growing culture suspension by continuous diffusion from a storage gel.Our results show that the controlled enzyme-based supply of glucose allows a glucose-limited growth to high cell densities of OD600 = 20 to 30 (corresponding to 6 to 9 g l-1 cell dry weight) without the external feed of additional compounds in shake flasks and 96-well plates. The final cell density can be further increased by addition of extra nitrogen during the cultivation. Production of a heterologous triosphosphate isomerase in E. coli BL21(DE3) resulted in 10 times higher volumetric product yield and a higher ratio of soluble to insoluble product when compared to the conventional production method. CONCLUSION: The novel EnBase method is robust and simple-to-apply for high cell density cultivation in shake flasks and microwell plates. The potential of the system is that the microbial growth rate and oxygen consumption can be simply controlled by the amount (and principally also by the activity) of the starch-degrading enzyme. This solves the problems of uncontrolled growth, oxygen limitation, and severe pH drop in shaken cultures. In parallel the method provides the basis for enhanced cell densities. The feasibility of the new method has been shown for 96-well plates and shake flasks and we believe that it can easily be adapted to different microwell and deepwell plate formats and shake flasks. Therefore EnBase will be a helpful tool especially in high throughput applications.

10.
Methods Mol Biol ; 1586: 127-137, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28470602

RESUMO

Expression of recombinant proteins in sufficient quantities is essential for protein structure-function studies. The most commonly used method for recombinant protein production is overexpression in E. coli cultures. However, producing high yields of functional proteins in E. coli can be a challenge in conventional shaken cultures. This is often due to nonoptimal growth conditions, which result in low cell yields and insoluble or incorrectly folded target protein. To overcome the shortcomings of shake flask cultivation, we present a cultivation method based on enzymatic glucose delivery. This system mimics the fed-batch principle used in bioreactor cultivations and provides high yields of biomass and recombinant proteins in shaken cultivations.


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Meios de Cultura/metabolismo , Escherichia coli/crescimento & desenvolvimento , Glucose/metabolismo , Proteínas Recombinantes/metabolismo , Técnicas de Cultura Celular por Lotes/instrumentação , Reatores Biológicos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glucose/administração & dosagem , Proteínas Recombinantes/genética , Regulação para Cima
11.
Microb Cell Fact ; 5: 8, 2006 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-16504107

RESUMO

BACKGROUND: Shake flasks are widely used because of their low price and simple handling. Many researcher are, however, not aware of the physiological consequences of oxygen limitation and substrate overflow metabolism that occur in shake flasks. Availability of a wireless measuring system brings the possibilities for quality control and design of cultivation conditions. RESULTS: Here we present a new wireless solution for the measurement of pH and oxygen from shake flasks with standard sensors, which allows data transmission over a distance of more than 100 metres in laboratory environments. This new system was applied to monitoring of cultivation conditions in shake flasks. The at-time monitoring of the growth conditions became possible by simple means. Here we demonstrate that with typical protocols E. coli shake flask cultures run into severe oxygen limitation and the medium is strongly acidified. Additionally the strength of the new system is demonstrated by continuous monitoring of the oxygen level in methanol-fed Pichia pastoris shake flask cultures, which allows the optimisation of substrate feeding for preventing starvation or methanol overfeed. 40 % higher cell density was obtained by preventing starvation phases which occur in standard shake flask protocols by adding methanol when the respiration activity decreased in the cultures. CONCLUSION: The here introduced wireless system can read parallel sensor data over long distances from shake flasks that are under vigorous shaking in cultivation rooms or closed incubators. The presented technology allows centralised monitoring of decentralised targets. It is useful for the monitoring of pH and dissolved oxygen in shake flask cultures. It is not limited to standard sensors, but can be easily adopted to new types of sensors and measurement places (e.g., new sensor points in large-scale bioreactors).

12.
J Biotechnol ; 117(4): 421-31, 2005 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-15876466

RESUMO

Lactic acid bacteria have an inefficient proteolytic system. Therefore, cultivation media which may have high protein content are usually supplemented with yeast extract or protein lysates (peptones). These additives might be conveniently replaced by in situ treatment of the cultivation medium with proteolytic enzymes or proteolytic microbes. Lactobacillus salivarius ssp. salicinius, a lactic acid bacterium species that can grow at high salt concentration, was used to ferment lactic acid in cheese whey (with 3 gl(-1) whey protein content) and lactose mother liquor (90 gl(-1) lactose, 9 gl(-1) proteins, 30 gl(-1) minerals). The contribution of protease enzymes or proteolytic microbes to acid production by lactobacilli was examined. Efficient conversion of lactose to lactic acid was obtained in the presence of additional proteolytic activity. Fastest acid production was obtained with the addition of protease enzymes. However, almost equally efficient acid production was obtained by treating the medium with Bacillus megaterium. The results show that fast and complete conversion of lactose to lactic acid can be obtained in dairy by-products without expensive additives.


Assuntos
Bacillus megaterium/metabolismo , Técnicas de Cultura de Células/métodos , Laticínios/microbiologia , Ácido Láctico/biossíntese , Lactobacillus/metabolismo , Peptídeo Hidrolases/metabolismo , Técnicas de Cocultura/métodos , Concentração de Íons de Hidrogênio , Resíduos Industriais/prevenção & controle , Ácido Láctico/química , Lactobacillus/classificação , Lactobacillus/crescimento & desenvolvimento , Sais/química
13.
Microb Cell Fact ; 4(1): 9, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15804347

RESUMO

SUMMARY: BACKGROUND: Escherichia coli induces the heat shock response to a temperature up-shift which is connected to the synthesis of a characteristic set of proteins, including ATP dependent chaperones and proteases. Therefore the balance of the nucleotide pool is important for the adaptation and continuous function of the cell. Whereas it has been observed in eukaryotic cells, that the ATP level immediately decreased after the temperature shift, no data are available for E. coli about the adenosine nucleotide levels during the narrow time range of minutes after a temperature up-shift. RESULTS: The current study shows that a temperature up-shift is followed by a very fast significant transient increase of the cellular ATP concentration within the first minutes. This increase is connected to a longer lasting elevation of the cellular respiration and glucose uptake. Also the mRNA level of typical heat shock genes increases within only one minute after the heat-shock. CONCLUSION: The presented data prove the very fast response of E. coli to a heat-shock and that the initial response includes the increase of the ATP pool which is important to fulfil the need of the cell for new syntheses, as well as for the function of chaperones and proteases.

14.
Microb Cell Fact ; 2(1): 2, 2003 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-12740045

RESUMO

BACKGROUND: Use of lactose-rich concentrates from dairy processes for the induction of recombinant gene's expression has not received much attention although they are interesting low cost substrates for production of recombinant enzymes. Applicability of dairy waste for induction of recombinant genes in Escherichia coli was studied. Clones expressing Lactobacillus phage muramidase and Lactobacillus alcohol dehydrogenase were used for the experiments. RESULTS: Shake flask cultivations in mineral salt medium showed that cheese whey or deproteinised whey induced gene expression as efficiently as IPTG (isopropyl-beta-D-thiogalactopyranoside) or pure lactose. Addition of yeast extract or proteolytically degraded whey proteins did not improve the recombinant protein yield. In contrast, addition of yeast extract to the well-balanced mineral salt medium decreased the product yield. Feeding with glycerol provided sufficient amount of easily assimilable carbon source during the induction period without preventing lactose intake and induction by lactose. High-cell-density fed-batch cultivations showed that product yields comparable to IPTG-induction can be achieved by feeding bacteria with a mixture of glycerol and concentrated whey permeate during the induction. CONCLUSION: Whey and concentrated whey permeate can be applied as an alternative inducer in recombinant high-cell-density fed-batch fermentations. The yield of the recombinant product was comparable to fermentations induced by IPTG. In low-cell-density shake flask experiments the yield was higher with whey or whey permeate than with IPTG.

15.
Biotechnol J ; 6(8): 1018-25, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21681961

RESUMO

Protein production and analysis in a parallel fashion is today applied in laboratories worldwide and there is a great need to improve the techniques and systems used for this purpose. In order to save time and money, a fast and reliable screening method for analysis of protein production and also verification of the protein product is desired. Here, a micro-scale protocol for the parallel production and screening of 96 proteins in plate format is described. Protein capture was achieved using immobilized metal affinity chromatography and the product was verified using matrix-assisted laser desorption ionization time-of-flight MS. In order to obtain sufficiently high cell densities and product yield in the small-volume cultivations, the EnBase® cultivation technology was applied, which enables cultivation in as small volumes as 150 µL. Here, the efficiency of the method is demonstrated by producing 96 human, recombinant proteins, both in micro-scale and using a standard full-scale protocol and comparing the results in regard to both protein identity and sample purity. The results obtained are highly comparable to those acquired through employing standard full-scale purification protocols, thus validating this method as a successful initial screening step before protein production at a larger scale.


Assuntos
Ensaios de Triagem em Larga Escala , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Cromatografia de Afinidade/métodos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Humanos , Biossíntese de Proteínas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
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