Selective flocculation of nucleic acids, lipids, and colloidal particles from a yeast cell homogenate by polyethyleneimine, and its scale-up

Milburn, Philip Thomas; Bonnerjea, J.; Hoare, Mike; Dunnill, P.

doi:10.1016/0141-0229(90)90070-7

Cited by 50 publications

(27 citation statements)

References 10 publications

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“…Most likely it is a form of flocculation, the destabilization of a suspension of colloids by the addition of a chemical agent known as a flocculant (Hocking et al, 1999). Consistent with our results, several studies have observed that polymer flocs bind large particles and proteins more readily than smaller ones (Agerkvist and Eriksson, 1993;Dimitrova et al, 2000;Juckes, 1971;Milburn et al, 1990;Salt et al, 1995;Tsoka et al, 2000). If it is the case that retroviruses are selectively incorporated into complexes of PB and CSC because of their large size, then it is likely that polymer complexation can be used to purify retroviruses from many other types of substances, in addition to free envelope proteins.…”

Section: Discussionsupporting

confidence: 94%

Complexation with chondroitin sulfate C and Polybrene rapidly purifies retrovirus from inhibitors of transduction and substantially enhances gene transfer

Landázuri

Doux

2005

Biotech & Bioengineering

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Using amphotropic retrovirus stocks produced by TELCeB6-A cells that encode the Escherichia coli lacZ gene, we found that complexation with chondroitin sulfate C (CSC) and Polybrene (PB) is an effective means to purify retrovirus. Virus stocks contained high levels of inhibitory activity that blocked amphotropic, but not ecotropic, retrovirus transduction. When virus stocks were brought to 80 microg/mL each of CSC and PB, complexes of CSC and PB formed. These complexes incorporated more than 70% of the virus particles but less than 0.4% of all other proteins and no detectable inhibitory activity. Purified virus transduced NIH 3T3 murine fibroblasts 21 to 186-fold more efficiently than virus that was not purified. In addition, virus purification significantly altered the dose response of transduction. When virus that had not been purified was used to transduce cells, the relationship between transduction and virus concentration was highly non-linear. In contrast, when purified virus was used, transduction increased monotonically and was linearly proportional to virus concentration, except when high doses of virus were used. Interestingly, when high doses of virus were used gene transfer reached a maximum plateau level, most likely because particle-associated amphotropic envelope proteins had saturated the cellular receptors for the virus. Our findings illustrate that retrovirus purification increases the maximum number of genes that can be transferred, reduces the amount of virus required to achieve a given level of gene transfer, and reduces uncertainties about the relationship between the amount of virus used and the number of genes transferred.

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Section: Discussionsupporting

confidence: 94%

Complexation with chondroitin sulfate C and Polybrene rapidly purifies retrovirus from inhibitors of transduction and substantially enhances gene transfer

Landázuri

Doux

2005

Biotech & Bioengineering

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“…The cell-free extract was mixed with equal volume of PEI solution (0.25 % polyethyleneimine MW 40K-60K, 6 % NaCl, 100 m Borax, pH 7.4) to remove lipids. [27] After centrifugation the supernatant was precipitated with 30 % ammonium sulfate. The resulting precipitate was collected after centrifugation and dissolved in potassium phosphate buffer (10 m, pH 7.2, 1 m DTT).…”

Section: Preparation Of Cell-free Extract and Purification Of Nitrilamentioning

confidence: 99%

Exploring the Synthetic Applicability of a Cyanobacterium Nitrilase as Catalyst for Nitrile Hydrolysis

et al. 2006

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The substrate specificity and synthetic applicability of the nitrilase from cyanobacterium Synechocystis sp. strain PCC 6803 have been examined. This nitrilase catalyzed the hydrolysis of both aromatic and aliphatic nitriles to the corresponding acids in high yields. Furthermore, the stereoselective hydrolysis of phenyl‐substituted β‐hydroxy nitriles to (S)‐enriched β‐hydroxy carboxylic acids and selective hydrolysis of α,ω‐dinitriles with five or less methylene groups to ω‐cyano carboxylic acids have been achieved. This suggested that nitrilase from Synechocystis sp. PCC 6803 could be a useful enzyme catalyst for the “green” nitrile hydrolysis. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

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“…The studies of precipitation embraced a range of agents, and the work [which was reviewed in Bell et al (1983)] has inevitably become increasingly concerned with precise descriptions of the engineering mechanisms (Ayazi Shamlou et al, 1994a). It also led to studies directed at more selective flocculation (Milburn et al, 1990) and to crystallization as a useful large-scale operation for proteins (Jacobsen et al, 1998). The natural corollary of this research has been a long-term concern with particle-liquid separation.…”

Section: Downstream Processingmentioning

confidence: 99%

UCL biochemical engineering

Shamlou¹,

Dunnill²,

Hoare³

et al. 1998

Biotechnol. Bioeng.

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Selective flocculation of nucleic acids, lipids, and colloidal particles from a yeast cell homogenate by polyethyleneimine, and its scale-up

Cited by 50 publications

References 10 publications

Complexation with chondroitin sulfate C and Polybrene rapidly purifies retrovirus from inhibitors of transduction and substantially enhances gene transfer

Complexation with chondroitin sulfate C and Polybrene rapidly purifies retrovirus from inhibitors of transduction and substantially enhances gene transfer

Exploring the Synthetic Applicability of a Cyanobacterium Nitrilase as Catalyst for Nitrile Hydrolysis

UCL biochemical engineering

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