RESUMEN
L-asparaginase is an essential drug used to treat acute lymphoid leukemia (ALL), a cancer of high prevalence in children. Several adverse reactions associated with L-asparaginase have been observed, mainly caused by immunogenicity and allergenicity. Some strategies have been adopted, such as searching for new microorganisms that produce the enzyme and applying protein engineering. Therefore, this work aimed to elucidate the molecular structure and predict the immunogenic profile of L-asparaginase from Penicillium cerradense, recently revealed as a new fungus of the genus Penicillium and producer of the enzyme, as a motivation to search for alternatives to bacterial L-asparaginase. In the evolutionary relationship, L-asparaginase from P. cerradense closely matches Aspergillus species. Using in silico tools, we characterized the enzyme as a protein fragment of 378 amino acids (39 kDa), including a signal peptide containing 17 amino acids, and the isoelectric point at 5.13. The oligomeric state was predicted to be a homotetramer. Also, this L-asparaginase presented a similar immunogenicity response (T- and B-cell epitopes) compared to Escherichia coli and Dickeya chrysanthemi enzymes. These results suggest a potentially useful L-asparaginase, with insights that can drive strategies to improve enzyme production.
Asunto(s)
Asparaginasa , Simulación por Computador , Penicillium , Asparaginasa/química , Asparaginasa/inmunología , Asparaginasa/metabolismo , Penicillium/inmunología , Penicillium/enzimología , Secuencia de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/inmunología , Proteínas Fúngicas/metabolismo , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/química , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/química , Humanos , Aspergillus/inmunología , Aspergillus/enzimología , Escherichia coli/genética , Dickeya chrysanthemi/enzimología , Dickeya chrysanthemi/inmunología , Modelos MolecularesRESUMEN
L-Asparaginase (L-ASNase) is an important enzyme used to treat acute lymphoblastic leukemia, recombinantly produced in a prokaryotic expression system. Exploration of alternatives production systems like as extracellular expression in microorganisms generally recognized as safe (such as Pichia pastoris Glycoswitch®) could be advantageous, in particular, if this system is able to produce homogeneous glycosylation. Here, we evaluated extracellular expression into Glycoswitch® using two different strains constructions containing the asnB gene coding for Erwinia chrysanthemi L-ASNase (with and without His-tag), in order to find the best system for producing the extracellular and biologically active protein. When the His-tag was absent, both cell expression and protein secretion processes were considerably improved. Three-dimensional modeling of the protein suggests that additional structures (His-tag) could adversely affect native conformation and folding from L-ASNase and therefore the expression and cell secretion of this enzyme.
Asunto(s)
Asparaginasa/genética , Clonación Molecular/métodos , Dickeya chrysanthemi/enzimología , Dickeya chrysanthemi/genética , Asparaginasa/química , Dickeya chrysanthemi/química , Expresión Génica , Genes Bacterianos , Glicosilación , Modelos Moleculares , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Tuberculosis (TB) resurged in the late 1980s and now kills approximately 3 million people a year. The reemergence of tuberculosis as a public health threat has created a need to develop new anti-mycobacterial agents. The shikimate pathway is an attractive target for herbicides and anti-microbial agents development because it is essential in algae, higher plants, bacteria, and fungi, but absent from mammals. Homologs to enzymes in the shikimate pathway have been identified in the genome sequence of Mycobacterium tuberculosis. Among them, the shikimate kinase I encoding gene (aroK) was proposed to be present by sequence homology. Accordingly, to pave the way for structural and functional efforts towards anti-mycobacterial agents development, here we describe the molecular modeling of M. tuberculosis shikimate kinase that should provide a structural framework on which the design of specific inhibitors may be based.