| dc.contributor.author |
Kotzia, GA |
en |
| dc.contributor.author |
Lappa, K |
en |
| dc.contributor.author |
Labrou, NE |
en |
| dc.date.accessioned |
2014-06-06T06:47:28Z |
|
| dc.date.available |
2014-06-06T06:47:28Z |
|
| dc.date.issued |
2007 |
en |
| dc.identifier.issn |
02646021 |
en |
| dc.identifier.uri |
http://dx.doi.org/10.1042/BJ20061708 |
en |
| dc.identifier.uri |
http://62.217.125.90/xmlui/handle/123456789/3609 |
|
| dc.subject |
Enzyme engineering |
en |
| dc.subject |
Hydrolase |
en |
| dc.subject |
L-asparaginase |
en |
| dc.subject |
Leukaemia |
en |
| dc.subject |
PEGylation |
en |
| dc.subject |
Poly(ethylene glycol) (PEG) |
en |
| dc.subject |
Proteolytic resistance |
en |
| dc.subject.other |
Ammonia |
en |
| dc.subject.other |
Bacteria |
en |
| dc.subject.other |
Biochemistry |
en |
| dc.subject.other |
Catalyst activity |
en |
| dc.subject.other |
Thermodynamic stability |
en |
| dc.subject.other |
Tumors |
en |
| dc.subject.other |
Enzyme engineering |
en |
| dc.subject.other |
Hydrolase |
en |
| dc.subject.other |
L-asparaginase |
en |
| dc.subject.other |
Leukaemia |
en |
| dc.subject.other |
PEGylation |
en |
| dc.subject.other |
Poly(ethylene glycol) (PEG) |
en |
| dc.subject.other |
Proteolytic resistance |
en |
| dc.subject.other |
Enzymes |
en |
| dc.subject.other |
arginine |
en |
| dc.subject.other |
asparaginase |
en |
| dc.subject.other |
glycine |
en |
| dc.subject.other |
histidine |
en |
| dc.subject.other |
hydrolase |
en |
| dc.subject.other |
lysine |
en |
| dc.subject.other |
macrogol |
en |
| dc.subject.other |
trypsin |
en |
| dc.subject.other |
amino acid sequence |
en |
| dc.subject.other |
article |
en |
| dc.subject.other |
enzyme stability |
en |
| dc.subject.other |
kinetics |
en |
| dc.subject.other |
matrix assisted laser desorption ionization time of flight mass spectrometry |
en |
| dc.subject.other |
nonhuman |
en |
| dc.subject.other |
Pectobacterium carotovorum |
en |
| dc.subject.other |
polyacrylamide gel electrophoresis |
en |
| dc.subject.other |
priority journal |
en |
| dc.subject.other |
protein degradation |
en |
| dc.subject.other |
protein engineering |
en |
| dc.subject.other |
protein expression |
en |
| dc.subject.other |
protein function |
en |
| dc.subject.other |
protein modification |
en |
| dc.subject.other |
protein purification |
en |
| dc.subject.other |
protein structure |
en |
| dc.subject.other |
sequence analysis |
en |
| dc.subject.other |
site directed mutagenesis |
en |
| dc.subject.other |
thermostability |
en |
| dc.subject.other |
Asparaginase |
en |
| dc.subject.other |
Base Sequence |
en |
| dc.subject.other |
DNA Primers |
en |
| dc.subject.other |
Enzyme Stability |
en |
| dc.subject.other |
Hydrolysis |
en |
| dc.subject.other |
Kinetics |
en |
| dc.subject.other |
Mutagenesis, Site-Directed |
en |
| dc.subject.other |
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization |
en |
| dc.subject.other |
Structure-Activity Relationship |
en |
| dc.subject.other |
Trypsin |
en |
| dc.subject.other |
Bacteria (microorganisms) |
en |
| dc.subject.other |
Pectobacterium carotovorum |
en |
| dc.title |
Tailoring structure-function properties of L-asparaginase: Engineering resistance to trypsin cleavage |
en |
| heal.type |
journalArticle |
en |
| heal.identifier.primary |
10.1042/BJ20061708 |
en |
| heal.publicationDate |
2007 |
en |
| heal.abstract |
Bacterial L-ASNases (L-asparaginases) catalyse the conversion of L-asparagine into L-aspartate and ammonia, and are widely used for the treatment of ALL (acute lymphoblastic leukaemia). In the present paper, we describe an efficient approach, based on protein chemistry and protein engineering studies, for the construction of trypsin-resistant PEGylated L-ASNase from Erwinia carotovora (EcaL-ASNase). Limited proteolysis of EcaL-ASNase with trypsin was found to be associated with a first cleavage of the peptide bond between Lys53 and Gly54, and then a second cleavage at Arg 206-Ser207 of the C-terminal fragment, peptide 54-327, showing that the initial recognition sites for trypsin are Lys53 and Arg206. Site-directed mutagenesis of Arg206 to histidine followed by covalent coupling of mPEG-SNHS [methoxypoly(ethylene glycol) succinate N-hydroxysuccinimide ester] to the mutant enzyme resulted in an improved modified form of EcaL-ASNase that retains 82% of the original catalytic activity, exhibits enhanced resistance to trypsin degradation, and has higher thermal stability compared with the wild-type enzyme. © 2007 Biochemical Society. |
en |
| heal.journalName |
Biochemical Journal |
en |
| dc.identifier.issue |
2 |
en |
| dc.identifier.volume |
404 |
en |
| dc.identifier.doi |
10.1042/BJ20061708 |
en |
| dc.identifier.spage |
337 |
en |
| dc.identifier.epage |
343 |
en |