Protein Purification: The Right Step at the Right Time

Bonnerjea, J.; Oh, Steven S.; Hoare, M.; Dunnill, P.

doi:10.1038/nbt1186-954

Cited by 147 publications

(38 citation statements)

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“…It is encouraging that, in precipitation and chromatography operations, 98% of the lipase can be recovered whereas, in the studies reported in literature, on the average only 75% recovery was achieved [28]. The purification factor depends strongly on the selectivity of each method and, therefore, this factor is, as expected, highest in the case of chromatography which is a high resolution technique, and lowest in foam fractionation which has a poor selectivity.…”

Section: Comparison Of Investigated Recovery Operationsmentioning

confidence: 55%

Comparison of selected methods for downstream processing in the production of bacterial lipase

et al. 1993

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Enzymes are of growing interest to the chemical and pharmaceutical industries as highly specific biocatalysts. An obstacle to a large-scale application of such proteins is the expenditure on recovery operations which can amount up to 80% of the total production costs. Therefore, it is necessary to investigate in detail the individual stages of downstream processing in order to obtain high product yields without loss of quality. After a survey of recovery operations and information on some special features of proteins which are relevant to downstream processing, this paper deals with the methods of precipitation, chromatography and foam fractionation. Several results obtained with a real, multicomponent system, namely the supernatant of lipase fermentation, are presented and compared with one another.

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Section: Comparison Of Investigated Recovery Operationsmentioning

confidence: 55%

Comparison of selected methods for downstream processing in the production of bacterial lipase

et al. 1993

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“…9±11 However, large-scale production of such proteins is limited because of their intracellular location. 6,12 Non-mechanical processes have also been proposed for selective cell permeabilization as alternatives to mechanical cell disruption. 8,13,14 These selective extraction methods are characterized by speci®c protein release, mainly for target proteins located within the periplasmic space or near the extracellular membrane.…”

Section: ±8mentioning

confidence: 99%

Penicillin acylase release from Escherichia coli cells by mechanical cell disruption and permeabilization

Fonseca

2002

J of Chemical Tech & Biotech

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The release of Penicillin acylase from Escherichia coli cells through mechanical cell disruption using high-pressure homogenization and sonication was studied. From these cell disruption processes, the enzyme activity was totally released although with low speci®c activities, 0.1±0.3 IU(mg prot) À1. Intracellular total soluble protein release was quanti®ed and modelled by a ®rst order kinetic model. The effect of the driving force for each mechanical method, namely acoustic power input and homogenization pressure, on the respective kinetic disruption constants was also analysed. The release of Penicillin acylase by cell permeabilization using osmotic shock was also evaluated. The effects of cell concentration, penicillin acylase activity in E coli cells, type of buffer, pH, hypertonic solution composition, temperature and time used for osmotic shock were evaluated. Using cold osmotic shock, highly selective penicillin acylase release was attained with speci®c enzyme activities of about 4 IU(mg prot)À1 and enzyme activity release yields higher than 90%. The high purity of the penicillin acylase was a consequence of the optimized differential enzyme release method which was validated by SDS gel electrophoresis.

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“…Due to its widespread applicability, its high resolution and capacity, as well as the simplicity and controllability of the method, it is probably the most frequently used chromatographic technique for the separation and purification of proteins, polypeptides, nucleic acids and other charged biomolecules [6]. As IEC is capable of separating species with very minor differences in properties, e.g.…”

Section: Introductionmentioning

confidence: 99%

Modeling the separation of small peptides by cation‐exchange chromatography

Harscoat–Schiavo¹,

Raminosoa²,

Ronat-Heit³

et al. 2010

J of Separation Science

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Cation-exchange chromatography was applied for the separation of short synthetic peptide standards, with various charges and hydrophobicities. The methodology to develop simple, reproducible and accurate models, based on physicochemical peptide properties, was described. A multilinear regression method was used for the calculation of the models, and descriptors were chosen according to the observed phenomena. The predictive and interpretative ability of the chromatographic models was evaluated considering cross-validated data (root mean-squared error of calibration, root mean-squared error, root mean-squared error of cross-validation and the Fisher ratio). Hydrophobic coefficients for amino acids were calculated with or without consideration of peptide sequences. A simple model, with only two parameters (charge and hydrophobic coefficient) was built. It enabled an accurate prediction of short peptide elution (up to nine residues). As the model was intended for further characterization of complex mixtures of unknown peptides, some mixtures were analyzed to investigate possible interactions between molecules. Peptides eluted in exactly the same pattern as when injected alone, supporting the use of these models for complex mixtures of small peptides.

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Protein Purification: The Right Step at the Right Time

Cited by 147 publications

References 1 publication

Comparison of selected methods for downstream processing in the production of bacterial lipase

Comparison of selected methods for downstream processing in the production of bacterial lipase

Penicillin acylase release from Escherichia coli cells by mechanical cell disruption and permeabilization

Modeling the separation of small peptides by cation‐exchange chromatography

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