Affinity chromatography of β‐glucuronidase

Harris, R. G.; Rowe, John J.; Stewart, Patrick D. Shaw; Williams, D. C.

doi:10.1016/0014-5793(73)80558-7

Cited by 33 publications

(10 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Among several reported affinity absorbents that had been used in GUS purification (saccharolactone, o-aminophenyl--D-glucuronide (Harris et al, 1973), and thiophenyl--D-glucuronide (Blanco and Nemoz, 1987)), saccharolactone was selected because of its commercial availability. For GUSD5, GUSD10, and GUSD15, two steps of affinity chromatography (saccharolactone immobilized on cross-linked 4% beaded agarose, Sigma, St. Louis, MO) and one more step of strong anion exchange chromatography (Q-sepharose fast flow, Sigma, St. Louis, MO) were used.…”

Section: Methodsmentioning

confidence: 99%

Genetic Engineering Strategies for Purification of Recombinant Proteins from Canola by Anion Exchange Chromatography: An Example of β-Glucuronidase

Zhang

Love²,

Jilka³

et al. 2001

Biotechnol. Prog.

View full text Add to dashboard Cite

The elution behavior of native canola proteins from different anion-exchange resins was determined. The elution profiles showed the potential for simplified recovery of acidic recombinant proteins from canola. When Q-sepharose fast flow was used, there were three optimal salt elution points at which a recombinant protein would have minimal contamination with native proteins. The feasibility of exploiting this advantage was examined for recovery of the acidic protein beta-glucuronidase (GUS/GUSD0 from the Escherichia coli gene) along with three polyaspartate fusions to the wild-type GUS. The fusions contained 5 (GUSD5), 10 (GUSD10), or 15 (GUSD15) aspartic acids fused to the C-terminus and were chosen to extend the elution time. The three fusions and the wild-type enzyme were produced in E. coli, purified, and added to canola extracts before chromatography. The equivalence of this spiking experiment to that of extracting a recombinant protein from transgenic canola was determined in a control experiment using transgenic canola expressing the wild-type enzyme. Behavior in the transgenic and spiked experiments was equivalent. GUSD0 eluted at the earliest optimal elution point; the addition of polyaspartate tails resulted in longer retention times and better selective recovery. If one assumes binding through a single fusion (the protein is a tetramer), there is a nearly linear shift in elution within the salt gradient of 17 mM per added charge up to 10, with a reduced increment from 10 to 15. The fusions and their enzymatic activity proved very stable in the canola extracts through 7 days in cold storage, providing flexibility in process scheduling.

show abstract

Section: Methodsmentioning

confidence: 99%

Genetic Engineering Strategies for Purification of Recombinant Proteins from Canola by Anion Exchange Chromatography: An Example of β-Glucuronidase

Zhang

Love²,

Jilka³

et al. 2001

Biotechnol. Prog.

View full text Add to dashboard Cite

show abstract

“…We used a fluorometric assay with 4-methylumbelliferyl ␤-d-glucuronide as the substrate (Gallagher, 1992) to determine the enzymatic activity from the clarified extracts. GUS protein was purified using ammonium sulfate precipitation (40%-50% saturation), followed by BioGel A1.5M size-exclusion chromatography (Bio-Rad), saccharo-1,4-lactone agarose (Sigma) affinity chromatography (Harris et al, 1973), and, finally, Mono-Q ion-exchange chromatography (Pharmacia). Purified GUS protein (50-100 g) was subjected to N-terminal amino acid sequence analysis using an Applied Biosystems model 470A protein sequencer.…”

Section: Enzyme Extraction and Analysismentioning

confidence: 99%

Use of Ubiquitin Fusions to Augment Protein Expression in Transgenic Plants1

et al. 1999

View full text Add to dashboard Cite

“…HARRIS et a!. (1973) andSKILLETER (1975) have confirmed the postulate that in the presence of triethyltin the chloride ion is able to become a competitive anion and thus displace substrate taken up into the matrix. However, SKILLETER (1975) concluded that the decrease of matrix substrate content is probably not the major cause of the greater sensitivity of oxidative phosphorylation to triethyltin in a predominantly chloride medium.…”

Section: Discussionmentioning

confidence: 87%

The Action of Triethyltin on the Respiration of Rat Brain Cortex Slices

Lock

1976

Journal of Neurochemistry

View full text Add to dashboard Cite

Abstract— Glucose oxidation by rat brain cortex slices is inhibited by low concentrations of triethyltin and this sensitive inhibition is shown to be chloride‐dependent. Pyruvate oxidation is only inhibited at high concentrations of triethyltin whether chloride is present or not. In contrast, the stimulation of both glucose and pyruvate oxidation observed with low concentrations of triethyltin prior to inhibition is chloride‐dependent. The results are discussed in relation to the chloride‐hydroxide exchange reaction known to be mediated across the inner mitochondrial membrane by triethyltin and other organo‐metals.

show abstract

Affinity chromatography of β‐glucuronidase

Cited by 33 publications

References 14 publications

Genetic Engineering Strategies for Purification of Recombinant Proteins from Canola by Anion Exchange Chromatography: An Example of β-Glucuronidase

Genetic Engineering Strategies for Purification of Recombinant Proteins from Canola by Anion Exchange Chromatography: An Example of β-Glucuronidase

Use of Ubiquitin Fusions to Augment Protein Expression in Transgenic Plants1

The Action of Triethyltin on the Respiration of Rat Brain Cortex Slices

Contact Info

Product

Resources

About