ABSTRACT
Inteins are intervening sequences that are transcribed and translated with flanking host protein sequences and then self-excised by protein splicing. Bi-functional inteins also contain a homing endonuclease responsible for their genetic mobility. The PRP8 intein, the most widespread among fungi, occurs in important pathogens such as Histoplasma capsulatum and Paracoccidioides brasiliensis, from the Ajellomycetaceae family. Herein, we describe the bi-functional PRP8 intein in two other Ajellomycetacean pathogens, Blastomyces dermatitidis and Emmonsia parva. Sequence analysis and experimental evidence suggest that the homing endonuclease from PbrPRP8 is inactive. The splicing activity of the PRP8 intein from the B. dermatitidis, E. parva and P. brasiliensis species complex was demonstrated in a non-native protein context in Escherichia coli. Since the PRP8 intein is located in a functionally essential nuclear protein, it can be considered a promising therapeutic target for anti-fungal drugs, because inhibition of intein splicing should inhibit proliferation of intein-containing pathogens.
Subject(s)
Blastomyces/enzymology , Chrysosporium/enzymology , Endonucleases/genetics , Endonucleases/metabolism , Inteins/genetics , Protein Splicing , Amino Acid Sequence , Blastomyces/genetics , Blastomyces/metabolism , Chrysosporium/genetics , Chrysosporium/metabolism , Cluster Analysis , Escherichia coli/genetics , Fungal Proteins/genetics , Fungal Proteins/metabolism , Molecular Sequence Data , Phylogeny , Sequence Alignment , Sequence AnalysisABSTRACT
The use of adsorption columns packed with ion-exchange resins for recovering, concentrating and purifying proteins is now widespread. The present work consists of a study on the dynamic behavior of adsorption columns that uses two kinds of adsorbents: a cationic and an anionic resin. A frontal analysis of the columns was performed with experimental data obtained from Fructozyme, a mixture of inulinase enzymes. The parameters of a Langmuir type of isotherm and adsorption kinetics were obtained from experimental tests in a batch system. A numerical technique based on orthogonal collocation and a fourth-order Runge-Kutta method was coupled with a nonlinear optimization method to predict the coefficients of the rate equations, which are fundamental for scale-up purposes.