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Improve the recombinant streptokinase production using fedbatch culture
V Mostra de
Pesquisa da PósGraduação
Juleane Lunardi1, 2, 4, Natasha Kuniechick2, 3, 4, Cláudia Paiva Nunes2, Luiz Augusto Basso1, 4,
Jocelei Chies2, Giandra Volpato2, Diógenes Santiago Santos1,3, 4.
1
Faculdade de Biociências, Programa de Pós-Graduação em Biologia Celular e Molecular, PUCRS,
2
Quatro G Pesquisa e Desenvolvimento Ltda,
3
Faculdade de Farmácia, PUCR,
4
Centro de Pesquisas em Biologia Molecular e Funcional, PUCR.
Streptokinase is a group of extracellular proteins produced by a variety of
streptococci beta-hemolytic strains, and is a plasminogen activator composed of 414 amino
acids with a molecular mass of 47 kDa. Unlike urokinase or tissue-type plasminogen
activator, that perform direct proteolysis, SK forms a high affinity equimolar complex with a
plasminogen (Kim et al., 2000). The resulting complex can convert plasminogen to plasmin,
the active protease that degrades fibrin in the blood clot (Wu et al., 1998). This enzyme has
been employed as a thrombolytic agent for the past 30 year (Wong et al., 1994) and it is now
widely used as a thrombolytic agent in the treatment of acute myocardial infarction, including
coronary thrombosis (Kim et al., 2000).
The low SK production yields from natural host and its pathogenicity are the main
reasons for exploration of recombinant DNA technology route for this important protein.
Escherichia coli is the most commonly used host for heterologous protein production and the
preferred method for increasing the concentration of heterologous recombinant protein, which
is proportional to both cell density and specific cellular product yield, is the fed-batch strategy
(Lee, 1996).
Fed-batch cultivation is an effective and simple method to increase the productivity of
a culture by increasing the cell concentration. Fed-batch cultures have been widely used for
recombinant protein production by Escherichia coli (Franz et al., 2005). During the preinduction phase, several feeding strategies have been used for increasing cell concentrations
in recombinant cultures, such as feeding based on a feedback control technique so as to keep
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the dissolved oxygen or pH constant (De Mare et al., 2005; Jeong et al., 2004) and linear or
exponential feed (Pal et al., 2001).
Streptokinase available in the Brazilian market is imported from South Korea,
increasing its costs for the government. Therefore, the objectives of this work are (a)
cultivation in bioreactor (first steps for the national production of this streptokinase), (b)
purification of the recombinant streptokinase and (c) measurement of its activity.
Streptococcus dysgalactiae subsp. equisimilis of the group C was obtained from a
Swab sample (from Dra. Ângela Castro - Departamento de Microbiologia da Universidade
Federal do Rio de Janeiro). Streptokinase gene was amplified by PCR using specific primers
containing NdeI e BamHI restriction sites. This amplicon was cloned into pCR-Blunt cloning
vector (Invitrogen) and the skc gene was subcloned into pET30a(+) expression vector
(Novagen) using NdeI e BamHI restriction enzymes. This plasmid was designated as
pET30a(+)-skc. E. coli BL21(DE3) cells were transformed with the pET30a(+)-skc
construction, and cultivated at 30°C in LB medium during 6 hours (protein expression
induced with 0.4 mM IPTG). Batch and fed-batch cultivations were carried out in a 2L
working volume stirred bioreactor (Biostat B plus, Sartorius Stedium, Germany) filled
initially with 1L of medium. The bioreactor was equipped with temperature, agitation,
aeration and pH controllers and two Rushton turbines with six flat-blades. Different fed-batch
media and four fed-batch strategy cultivations were tested, two with feedback control (DOstat and pH-stat) and two without feedback control (linear and exponential). After defining the
better fed-batch media and strategy, different induction times were studied.
In all experiments, protein analysis was carried out by SDS-PAGE electrophoresis.
The concentration of SK will be measured according to Bradford et al. (1976) and
densitometry analysis (GS-700 Bio-Rad). The experiments of the purification are being
conducted in GE Pharmacia Äkta Purifier. SK activity will be assayed by the method of
Pratap et al. (1996) using chromozyme PL (Roche) as an artificial substrate.
The best expression of the SKC was obtained in LB culture medium at 30ºC for E. coli
BL21(DE3) strain. The fed-batch cultivation defined was a feeding medium MgSO4 (25g/L)
and glucose (400g/L). The maximal biomass production and protein expression were achieved
using linear feeding strategy at 12h of induction, increasing the biomass and cell productivity
of approximately 8 fold and 7 fold, respectively, when compared to shaker cultivation.
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Referências
BRADFORD, M. M. A rapid and sensitive method for the quantization of microgram quantities of protein
utilizing the principle of protein-dye binding. Analytical Biochemistry. Vol. 7 (1976), pp. 248-254.
DE MARE, L., VELUT, S., LEDUNG, E., CIMANDER, C., NORRMAN, B., NORDBERG KARLSSON,
E., HOLST, O., HAGANDER, P. A cultivation technique for E. coli fedbatch cultivations operating close to
the maximum oxygen transfer capacity of the reactor. Biotechnology Letters. Vol. 27 (2005), pp. 983–990.
FRANZ, C., JURGEN, K., FLORENTINA, P., KARL, B. Sensor combination and chemometric modeling
for improved process monitoring in recombinant E. coli fed-batch cultivations. Journal of Biotechnology.
Vol. 120 (2005), pp. 183–196.
JEONG, K.J., CHOI, J.H., YOO, W.M., KEUM, K.C., YOO, N.C., LEE, S.Y., SUNG, M.H. Constitutive
production of human leptin by fed-batch culture of recombinant rpoS-Escherichia coli. Protein and
Expression Purification. Vol. 36 (2004), pp. 150–156.
KIM, D.M., LEE, S.J., KIM, I.C., KIM, S.T., BYUN, S.M. Asp41-His48 region of streptokinase is
important in binding to a substrate plasminogen. Thrombolytic Resources. Vol. 99 (2000), pp. 93-98.
LEE, S.Y. High cell-density culture of Escherichia coli. Trends of Biotechnology. Vol. 1996, 14, 98-105.
PAL, Y., GUPTA, J.C., MUKHERJEE, K.J. Optimizing recombinant protein expression in the T7 system
under the control of the proUp promoter. Biotechnology Letters. Vol. 23 (2001), pp.41–46.
PRATAP, J., KAUR, J., RAJAMOHAN, G., SINGH, D., DIKSHIT, K.L. Role of N-terminal domain of
streptokinase in protein transport. Biochemical and Biophysical Research Communications. Vol. 227
(1996), pp. 303-310.
WONG, S.L., YE, R., NATHOO, S. Engineering and production of streptokinase in a Bacillus subtilis
expression-secretion system. Applied Environmental Microbiology. Vol. 60 (1994), pp. 517-523.
WU, X.C., YE, R., DUAN, Y., WONG, S.L. Engineering of plasmin-resistant forms of streptokinase and
their production in Bacillus subtilis: Streptokinase with longer functional half-life. Applied Environmental
Microbiology. Vol. 64 (1998), pp. 824-829.
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