Transcript Characterization and protein engineering of L

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
High ASNS
Low ASNS
ASNS-negative
Adapted from 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
15M
58M
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