A novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein

Hedhammar, My; Stenvall, Maria; Lönneborg, Rosa; Nord, Olof; Sjölin, Olle; Brismar, Hjalmar; Uhlén, Mathias; Ottosson, Jenny; Hober, Sophia

doi:10.1016/j.jbiotec.2005.03.024

Cited by 33 publications

(32 citation statements)

References 32 publications

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“…Total protein was determined using the BCA assay (Pierce Biotechnology, Inc., Rockford, IL). Monitoring GFP production by flow cytometry and fluorometry is linear in relation to the amount of protein produced down to mmol L À1 concentrations (Hedhammar et al, 2005;Remans et al, 1999), is non-destructive to GFP-producing cells, can measure GFP production in single cells, and can differentiate cells based on the degree of their GFP expression.…”

Section: Green Fluorescent Protein Concentrationmentioning

confidence: 99%

Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm

O’Connell

Niu

Gilbert

2006

Biotech & Bioengineering

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Escherichia coli has been widely used for heterologous protein production (HPP). To determine whether a biofilm environment could benefit E. coli HPP using high copy number plasmids, we compared plasmid maintenance and HPP by E. coli ATCC 33456 containing plasmid pEGFP (a pUC family vector) cultivated in biofilms and in suspended culture. Cells were grown with or without antibiotic selective pressure in flow cells or chemostats for up to 6 days. In biofilms, antibiotic selective pressure increased the plasmid copy number (PCN), but by 144 h, biofilms grown in antibiotic-free media had comparable plasmid concentrations. In the chemostat, the PCN declined steadily, although 100 ppm ampicillin in the medium slowed the rate of plasmid loss. Production of green fluorescent protein (GFP), a representative heterologous protein, was quantified by flow cytometry. In biofilms, at ampicillin concentrations >or=33 ppm, strongly fluorescent cells comprised more than half of the population by 48 h. In the chemostat, more than 50% of the population was non-fluorescent by 48 h in media containing 100 ppm ampicillin, and strongly fluorescent cells were <10% of the population. Biofilm structure was determined by confocal microscopy. Maximum biofilm thickness ranged from 30 to 45 microns, with no significant changes in biofilm structure after 48 h. Plasmid multimer percentages were similar to inocula for cells cultivated in either biofilms or the chemostat. The results indicate that the biofilm environment enhanced both plasmid maintenance and cellular GFP concentrations, and that low levels of antibiotic increased the beneficial effect.

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Section: Green Fluorescent Protein Concentrationmentioning

confidence: 99%

Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm

O’Connell

Niu

Gilbert

2006

Biotech & Bioengineering

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“…Load the cleavage mixture onto a 1 mL HiTrap S column previously equilibrated with 5 CV of Buffer A s . [ 14 ]. We typically use the Escherichia coli ( E. coli ) strain BL21 (DE3) (Novagen) for production.…”

Section: Purifi Cation Of Soluble Z Basic Fusion Proteinsmentioning

confidence: 99%

Zbasic: A Purification Tag for Selective Ion-Exchange Recovery

Hedhammar

Nilvebrant

Hober

2014

Methods in Molecular Biology

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A positively charged protein domain, denoted Zbasic, can be used as a general purification tag for purification of recombinantly produced target proteins by cation-exchange chromatography. The Zbasic domain is constructed from the Protein A-derived domain and engineered to be highly charged, which allows selective capture on a cation exchanger at physiological pH values. Moreover, Zbasic is selective also under denaturing conditions and can be used for purification of proteins solubilized from inclusion bodies. Zbasic can then be used as a flexible linker to the cation-exchange resin and thereby allow solid-phase refolding of the target protein.Herein, protocols for purification of soluble Zbasic-tagged fusion proteins, as well as for integrated purification and solid-phase refolding of insoluble fusion proteins, are described. In addition, a procedure for enzymatic tag removal and recovery of native target protein is outlined.

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“…Study of light scattering by granulated biological cells is motivated by several empirical techniques that use the dependence of light scattering signals measured by flow cytometer on the characteristics of the granules [14][15][16][17]. All these techniques are successfully used in practice but lack a rigorous theoretical foundation.…”

Section: Introductionmentioning

confidence: 99%

“…All these techniques are successfully used in practice but lack a rigorous theoretical foundation. For instance, production of the inclusion bodies consisting of protein synthesized by E. coli cells was found to correlate with forward scattering [16,17], which can be qualitatively explained by the increase of total amount of matter that scatters light inside the cells. Probably, the most fascinating application of light scattering to characterize granulated cells are the results of de Grooth, Terstappen and coworkers [14,18,19], who showed that neutrophils and eosinophils can be discriminated by their depolarized side scattering intensity I ⊥ .…”

Section: Introductionmentioning

confidence: 99%

Discrimination of granulocyte subtypes from light scattering: theoretical analysis using a granulated sphere model

Yurkin¹,

Semyanov²,

Maltsev³

et al. 2007

Opt. Express

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Discrimination of granulocyte subtypes from light scattering: theoretical analysis using a granulated sphere model Yurkin, M.A.; Semyanov, K.A.; Maltsev, V.P.; Hoekstra, A.G. General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Virology and Biotechnology "VECTOR", Koltsovo, 630559, Novosibirsk Region, Russia *myurkin@science.uva.nl Abstract: We perform extensive simulations of light scattering by a granulated sphere in the size and refractive index range of human granulated leucocytes using the discrete dipole approximation. We calculate total and depolarized side scattering signals as a function of the size and refractive indices of cell and granules, and the granule volume fraction. Using typical parameters derived from the literature data on granulocyte morphology, we show that differences between experimentally measured signals of two granulocyte subtypes can be explained solely by the difference in their granule sizes. Moreover, the calculated depolarization ratio quantitatively agrees with experimental results. We also use the Rayleigh-Debye-Gans approximation and its second order extension to derive analytical expressions for side scattering signals. These expressions qualitatively describe the scaling of signals with varying model parameters obtained by rigorous simulations, and even lead to quantitative agreement in some cases. Finally, we show and discuss the dependence of extinction efficiency and asymmetry parameter on size and volume fraction of granules.

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A novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein

Cited by 33 publications

References 32 publications

Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm

Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm

Zbasic: A Purification Tag for Selective Ion-Exchange Recovery

Discrimination of granulocyte subtypes from light scattering: theoretical analysis using a granulated sphere model

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