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Expression patterns of recombinant asparaginase in different E.
coli strains
V Mostra de
Pesquisa da PósGraduação
Gustavo Roth1,2,3, Giandra Volpato3, Christiano Ev Neves2, Gaby Renard3, Luiz Augusto Basso2,
Jocelei Maria Chies3, Diógenes Santiago Santos2,3
1
Curso de Pós-Graduação em Biologia Celular e Molecular, Faculdade de Biociências, PUCRS, 2Centro de
Pesquisa em Biologia Molecular e Funcional, Instituto Nacional de Ciência e Tecnologia em Tuberculose,
3
Quatro G Pesquisa e Desenvolvimento Ltda.
Abstract
L-Asparaginase enzymes (L-Asparagine amidohydrolase; EC 3.5.1.1) catalyze the
hydrolysis of L-Asparagine (L-Asn) to L-Aspartate (L-Asp) and ammonia (NH3), and to a
lesser extent the hydrolysis of L-Glutamine (L-Gln) to L-Glutamate (L-Glu) (Wang et al.,
2001). Two types of bacterial L-asparaginases have been identified: type I and type II
(Campbell et al., 1967). Type II L-asparaginases, in particular, present tumor inhibitory
activity and have thus been extensively studied (Cedar and Schwartz, 1968).
Tumor-inhibitory L-asparaginases have been isolated from a number of bacterial
sources. However, their glutaminase activity has been implicated in causing serious side
effects. Fortunately, L-asparaginase II from Erwinia carotovora, which was used in this work,
has significantly lower glutaminase activity and may represent an important alternative
therapy (Hawkins et al., 2004; Krasotkina et al., 2004).
The study of the expression patterns of recombinant asparaginase in different E. coli
strains, which is the aim of this work, is an important step for later commercial production.
Among the alternatives for the production of this enzyme, we can name the direct isolation
from the microorganism, or the production of the enzyme through recombinant technology in
genetically modified E. coli. Nowadays, E. coli is the most well-studied microorganism. This
knowledge allows some easiness in the manipulation of the bacteria and the production of
heterologous proteins (Lima, 2004). Genetic manipulation allows the creation of mutant
strains with specific characteristics; this differentiation is favorable for better recombinant
protein expression.
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After amplification of the asparaginase gene from Erwinia carotovora, the gene was
cloned into the expression vector pET30a(+) and used to transform different E. coli strain by
electroporation. As control the plasmid lacking the asparaginase gene was used. The E. coli
strains used were: BL21 (DE3) NH, BL21 (DE3) Star, C41 (DE3), C43 (DE3), Rosetta
(DE3), and BL21 (DE3).
The TB culture medium was used at 37ºC and 30ºC, and was inoculated to an OD600nm
of 0,1. The optical density was monitored at each hour. Cultures were induced, or not, with 1
mM IPTG after reaching OD600nm 0.4 - 0.6. For control the E. coli strain was transformed with
the plasmid lacking the asparaginase gene. For expression analysis 1 mL of the cultures were
submitted to electrophoresis on polyacrylamide gels (SDS-PAGE). Samples were collected at
3, 6, 9 and 24 hours after induction. In order to separate the soluble and insoluble fractions,
the samples were centrifuged at 13,000 rpm for 3 min and frozen at -20ºC.
All E. coli strains tested showed the superexpression of the asparaginase at 37oC. At
the moment, the cultures are being tested at 30oC in order to observe if the temperature will
influence in the expression of the protein.
These studies are important for the scaling-up process in the bioreactor, aiming the
national production of this enzyme.
Referências
CAMPBELL, H.A., MASHBURN, L.T., BOYSE, E.A., OLD, L.J., Two L-asparaginases from Escherichia coli
B. Their separation, purification, and antitumor activity. Biochemistry. Vol. 6, (1967), pp. 721-730.
CEDAR, H., SCHWARTZ, J.H. Production of L-asparaginase II by Escherichia coli. Journal of Bacteriology.
Vol. 96, (1968), pp. 2043-2048.
HAWKINS, D. S.; PARK, J. R.; THOMSON, B. G.; FELGENHAUER, J. L.; HOLCENBERG, J. S.;
PANOSYAN, E. H.; AVRAMIS, V. I. Asparaginase pharmacokinetics after intensive polyethylene glycolconjugated L-asparaginase therapy for children with relapsed acute lymphoblastic leukemia. Clinical Cancer
Research; v. 10, n. 16 (2004), p. 5335-5341.
KRASOTKINA, J.; BORISOVA, A. A.; GERVAZIEV, Y. V.; SOKOLOV, N. N. One-step purification and
kinetic properties of the recombinant L-asparaginase from Erwinia carotovora. Biotechnology and Applied
Biochemistry; v. 39, n. 2, (2004), p. 215-221.
LIMA, W. J. N. Produção de Proteínas Rescombinantes Utilizando Escherichia coli em Cultivos em Alta
Densidade. Tese de Doutorado, Unicamp, 181, 2004.
WANG, Y., QIAN, S., MENG, G., ZHANG, S, Cloning and expression of L-Asparaginase gene in Escherichia
coli. Applied Biochemistry and Biotechnology. Vol. 95, (2001), pp. 93-101.
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