English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Tools annual

Global: Zendy Free Plan

Global: Zendy Tools monthly

Global: Zendy Plus monthly

Abstract Background Transformed cells of <it>Escherichia coli DH5-α</it> with pGFPuv, induced by IPTG (isopropyl-β-d-thiogalactopyranoside), express the green fluorescent protein (<it>gfp</it><it>uv</it>) during growth phases. <it>E. coli</it> subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-phase partitioning extraction (TPP) method were compared to the direct application of TPP to the same culture of <it>E. coli</it> on releasing <it>gfp</it><it>uv</it> from the over-expressing cells. Material and Methods Cultures (37°C/100 rpm/ 24 h; μ = 0.99 h-1 - 1.10 h-1) of transformed (<it>pGFP</it>) <it>Escherichia coli DH5-α</it>, expressing the green fluorescent protein (<it>gfp</it><it>uv</it>, absorbance at 394 nm and emission at 509 nm) were sonicated in successive intervals of sonication (25 vibrations/pulse) to determine the maximum amount of <it>gfp</it><it>uv</it> released from the cells. For selective permeation, the transformed previously frozen (-75°C) cells were subjected to three freeze/thaw (-20°C/ 0.83°C/min) cycles interlaid by sonication (3 pulses/ 6 seconds/ 25 vibrations). The intracellular permeate with <it>gfp</it><it>uv</it> in extraction buffer (TE) solution (25 mM Tris-HCl, pH 8.0, 1 mM β-mercaptoethanol β-ME, 0.1 mM PMSF) was subjected to the three-phase partitioning (TPP) method with <it>t</it>-butanol and 1.6 M ammonium sulfate. Sonication efficiency was verified on the application to the cells previously treated by the TPP method. The intra-cell releases were mixed and eluted through methyl HIC column with a buffer solution (10 mM Tris-HCl, 10 mM EDTA, pH 8.0). Results The sonication maximum released amount obtained from the cells was 327.67 μg <it>gfp</it><it>uv</it>/mL (20.73 μg <it>gfp</it><it>uv</it>/mg total proteins – BSA), after 9 min of treatment. Through the selective permeation by three repeated freezing/thawing/sonication cycles applied to the cells, a close content of 241.19 μg <it>gfp</it><it>uv</it>/mL (29.74 μg <it>gfp</it><it>uv</it>/mg BSA) was obtained. The specific mass range of <it>gfp</it><it>uv</it> released from the same cultures, by the three-phase partitioning (TPP) method, in relation to total proteins, was higher, between 107.28 μg/mg and 135.10 μg/mg. Conclusions The selective permeation of <it>gfp</it><it>uv</it> by freezing/thawing/sonication followed by TPP separation method was equivalent to the amount of <it>gfp</it><it>uv</it> extracted from the cells directly by TPP; although selective permeation extracts showed better elution through the HIC column.</p

Selective permeation and organic extraction of recombinant green fluorescent protein (gfpuv) from Escherichia coli.