Glycerol Increases the Yield and Activity of Human Phenylalanine Hydroxylase Mutant Enzymes Produced in a Prokaryotic Expression System

Leandro, Paula; Lechner, Maria Celeste; Almeida, Isabel Tavares de; Konecki, David

doi:10.1006/mgme.2001.3172

Cited by 49 publications

(27 citation statements)

References 16 publications

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“…For instance, proline protected chicken liver fatty acid synthase , chicken egg lysozyme (Samuel et al, 2000), and rabbit skeletal muscle creatine phosphokinase (Meng et al, 2001) against aggregation. A wide range of osmolytes can restore mutant protein folding defects in cystathionine beta synthase (Singh et al, 2007) and phenylalanine hydroxylase (Leandro et al, 2001). However, other researchers reported that osmolytes rather potentiate misfolding or have no effect on protein aggregation.…”

Section: Stabilization And/or Destabilization By Osmolytes Modulates mentioning

confidence: 97%

“…Protecting osmolytes stabilize proteins against denaturation without altering their functional activities (Taneja and Ahmad, 1994;Anjum et al, 2000;Xie and Timasheff, 1997a,b,c;Santoro et al, 1992;Foord and Leatherbarrow, 1998;Kim et al, 2003;Myers and Jakoby, 1975). Osmolytes also help to stabilize unstable proteins and/or refold misfolded proteins (Leandro et al, 2001;Leandro and Gomes, 2008;Singh et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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Protein and DNA destabilization by osmolytes: The other side of the coin

et al. 2011

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Section: Stabilization And/or Destabilization By Osmolytes Modulates mentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

Protein and DNA destabilization by osmolytes: The other side of the coin

et al. 2011

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“…The variability in the specific activities observed in different soluble enzymes when produced under different environmental conditions strongly supports this presumption. 7,[15][16][17][18] How the amyloidal deposition of conformationally hybrid, misfolded but functional proteins is regulated by the cell is not known.…”

Section: Introductionmentioning

confidence: 99%

Divergent Genetic Control of Protein Solubility and Conformational Quality in Escherichia coli

García‐Fruitós

Martínez-Alonso

González-Montalbán

et al. 2007

Journal of Molecular Biology

102

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“…Glycerol can improve protein stability and increase the specific activity of enzymes (Bradbury and Jakoby, 1972;Leandro et al, 2001). Therefore, we investigated the effect of the addition of glycerol following protein purification.…”

Section: Optimization Of Xxt2 Expressionmentioning

confidence: 99%

Enzymatic Activity of Xyloglucan Xylosyltransferase 5

et al. 2016

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ORCID IDs: 0000-0002-5107-9009 (R.F.); 0000-0001-6938-6923 (O.A.Z.).Xyloglucan, the most abundant hemicellulosic component of the primary cell wall of flowering plants, is composed of a b-(1,4)-glucan backbone decorated with D-xylosyl residues. Three xyloglucan xylosyltransferases (XXTs) participate in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana). Two of these, XXT1 and XXT2, have been shown to be active in vitro, whereas the catalytic activity of XXT5 has yet to be demonstrated. By optimizing XXT2 expression in a prokaryotic system and in vitro activity assay conditions, we demonstrate that nonglycosylated XXT2 lacking its cytosolic amino-terminal and transmembrane domain displays high catalytic activity. Using this optimized procedure for the expression of XXT5, we report, to our knowledge for the first time, that recombinant XXT5 shows enzymatic activity in vitro, although at a significantly slower rate than XXT1 and XXT2. Kinetic analysis showed that XXT5 has a 7-fold higher K m and 9-fold lower k cat compared with XXT1 and XXT2. Activity assays using XXT5 in combination with XXT1 or XXT2 indicate that XXT5 is not specific for their products. In addition, mutagenesis experiments showed that the in vivo function and in vitro catalytic activity of XXT5 require the aspartate-serine-aspartate motif. These results demonstrate that XXT5 is a catalytically active xylosyltransferase involved in xylosylation of the xyloglucan backbone.

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Glycerol Increases the Yield and Activity of Human Phenylalanine Hydroxylase Mutant Enzymes Produced in a Prokaryotic Expression System

Cited by 49 publications

References 16 publications

Protein and DNA destabilization by osmolytes: The other side of the coin

Protein and DNA destabilization by osmolytes: The other side of the coin

Divergent Genetic Control of Protein Solubility and Conformational Quality in Escherichia coli

Enzymatic Activity of Xyloglucan Xylosyltransferase 5

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