Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Characterization and protein engineering of L
Document related concepts
Transcript
Characterization and protein engineering of Lasparaginase 1 from Saccharomyces cerevisiae to evaluate its use as biopharmaceutical Prof. Dr. Gisele Monteiro Department of Biochemical and Pharmaceutical Technology Faculty of Pharmaceutical Sciences University of São Paulo - USP Leukemia • Blood cancer • Prevalence in childhood – 80% cases • Treatment – chemotherapy and Lasparaginase Complete remission and cure Discovery of L-asparaginase (L-ASNase) Kidd 1953 Catalytic action Antitumor activity Wai Kin Chan et al. Blood 2014;123:3596-3606 ©2014 by American Society of Hematology Source of L-ASNase L-ASNase II Escherichia coli Erwinia chrysanthemi KM 15M 58M kcat 2,7x103 s-1 23,8x103 s-1 5,1x105 M-1 s-1 4,1x105M-1 s-1 200U/mg 120U/mg 6.000UI/m2 25.000UI/m2 kcat/KM Specific activity Dose in the treatment • Allergic reactions • Hyperammonemia • Immune reactions – Antibodies and proteases • Silence inactivation Problems Objective • Test the potencial of ScASNaseI as biopharmaceutical to treat Acute lymphoblastic leukemia - ALL Results No glutaminase activity detected K0,5 mM ScASNase1 0.075 ± 0.027 Vmax μmol/min 0.0083 ± 0,0002290 Kcat s-1 Kcat/ S0,5 M/s nH 35 ± 0.9 4.65x105 2.2 ± 0.3 Amino acids – catalytic site Mutant K215A T141A T64A Y78A Specific Activity (U/mg) 0,024 0,043 0,1 0,172 Residual Activity % 0,012 0,022 0,051 0,088 Results • Circular Dichroisms Asp1-WT T64A K215A Prof. Dr. Marcos Oliveira - Unesp T141A Y78A Cytotoxicity assay – MOLT-4 leukemia cells ScASNaseI Kidrolase EcASNaseII Concluding Remarks • ScASNase has potential to be a new biopharmaceutical to treat ALL • High specific activity • No glutaminase activity • Eukaryotic protein • Allosteric enzyme • High substrate affinity • Antitumor activity Thanks to authors Financial support. Grants 2013/08617-7 and 2013/16685-2
