Expression in Escherichia coli and Purification of Microbial Enzymes for the Production of Enzymatic Analytical Kits to Measure Relevant Metabolites
Patrícia I. P. Ponte
NZYTech - Genes and Enzymes, Estrada do Paço do Lumiar, Campus Lumiar, Edifício E, R/C, 1649-038 Lisboa, Portugal
Catarina I. P. D. Guerreiro
NZYTech - Genes and Enzymes, Estrada do Paço do Lumiar, Campus Lumiar, Edifício E, R/C, 1649-038 Lisboa, Portugal
Vânia Fernandes
NZYTech - Genes and Enzymes, Estrada do Paço do Lumiar, Campus Lumiar, Edifício E, R/C, 1649-038 Lisboa, Portugal
André T. Fernandes
NZYTech - Genes and Enzymes, Estrada do Paço do Lumiar, Campus Lumiar, Edifício E, R/C, 1649-038 Lisboa, Portugal
Carlos M. G. A. Fontes
NZYTech - Genes and Enzymes, Estrada do Paço do Lumiar, Campus Lumiar, Edifício E, R/C, 1649-038 Lisboa, Portugal and CIISA, Faculdade de Medicina Veterinária, ULisboa, Avenida da Universidade Técnica, Pólo Universitário do Alto da Ajuda, 1300-477 Lisboa, Portugal
Luís M. A. Ferreira
NZYTech - Genes and Enzymes, Estrada do Paço do Lumiar, Campus Lumiar, Edifício E, R/C, 1649-038 Lisboa, Portugal and CIISA, Faculdade de Medicina Veterinária, ULisboa, Avenida da Universidade Técnica, Pólo Universitário do Alto da Ajuda, 1300-477 Lisboa, Portugal
José A. M. Prates *
NZYTech - Genes and Enzymes, Estrada do Paço do Lumiar, Campus Lumiar, Edifício E, R/C, 1649-038 Lisboa, Portugal and CIISA, Faculdade de Medicina Veterinária, ULisboa, Avenida da Universidade Técnica, Pólo Universitário do Alto da Ajuda, 1300-477 Lisboa, Portugal
*Author to whom correspondence should be addressed.
Abstract
Aim: The present study describes the procedure for the isolation, cloning, expression and purification of eight different microbial enzymes required for the assembly of a comprehensive range of enzymatic test kits.
Methodology: The genes encoding the enzymes were isolated through Polymerase Chain Reaction or synthesized In vitro and inserted into a variety of prokaryotic expression vectors. The resulting recombinant proteins were expressed at high levels in Escherichia coli cells and purified following different chromatographic approaches. The catalytic activity of each individual enzyme was determined.
Results: Eight enzymes (aspartate aminotransferase, EC 2.6.1.1; citrate synthase, EC 2.3.3.1; glucose-6-phosphate dehydrogenase, EC 1.1.1.49; hexokinase, EC 2.7.1.2; L-malate dehydrogenase, EC 1.1.1.37; D-malate dehydrogenase, EC 1.1.1.83; and glucose-6-phosphate isomerase, EC 5.3.1.9) were engineered to contain a His6-tag, cloned and expressed in E. coli BL21(DE3) strain, which was grown in auto-induced LB medium. Recombinant proteins expression, solubility and yields were analyzed. All recombinant proteins were expressed in soluble form with expression induction at 37°C, although the induction of expression of citrate synthase at 20°C resulted in higher levels of soluble protein. The recombinant proteins were captured by Immobilized Metal-Affinity Chromatography and polished by gel filtration. All proteins were purified from most E. coli contaminants (>95%) and were obtained at high concentrations (2-17 g/L). Specific activities varied from 2.1 to 1220 U/mg, depending on the enzyme.
Conclusion: The methodology presented here is suitable for the expression and recovery of highly purified recombinant enzymes for the development of enzymatic analytical kits, with relevant agricultural, biomedical and industrial applications.
Keywords: Recombinant enzymes, analytical kits, protein expression, Escherichia coli