RESUMEN
Sixteen of 22 low molecular weight integral membrane proteins from Mycobacterium tuberculosis with previously poor or undetectable levels of expression were expressed in Escherichia coli as fusions with both the maltose binding protein (MBP) and a His(8)-tag. Sixty-eight percent of targeted proteins were expressed in high yield (>30 mg/L) in soluble and/or inclusion body form. Thrombin cleavage of the MBP fusion protein was successful for 10 of 13 proteins expressed as soluble proteins and for three proteins expressed only as inclusion bodies. The use of autoinduction growth media increased yields over Luria-Bertani (LB) growth media in 75% of the expressed proteins. Expressing integral membrane proteins with yields suitable for structural studies from a set of previously low and non-expressing proteins proved highly successful upon attachment of the maltose binding protein as a fusion tag.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Escherichia coli/metabolismo , Expresión Génica , Proteínas de la Membrana/metabolismo , Mycobacterium tuberculosis/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Clonación Molecular , Escherichia coli/genética , Histidina/química , Cuerpos de Inclusión/metabolismo , Proteínas de Unión a Maltosa , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Datos de Secuencia Molecular , Peso Molecular , Plásmidos , Reacción en Cadena de la Polimerasa , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , SolubilidadRESUMEN
Many of the protein fusion systems used to enhance the yield of recombinant proteins result in the addition of a small number of amino acid residues onto the desired protein. Here, we investigate the effect of short (three amino acid) N-terminal extensions on the equilibrium denaturation and kinetic folding and unfolding reactions of the FK506-binding protein (FKBP) and compare the results obtained with data collected on an FKBP variant lacking this extension. Isothermal equilibrium denaturation experiments demonstrated that the N-terminal extension had a slight destabilizing effect. NMR investigations showed that the N-terminal extension slightly perturbed the protein structure near the site of the extension, with lesser effects being propagated into the single alpha-helix of FKBP. These structural perturbations probably account for the differential stability. In contrast to the relatively minor equilibrium effects, the N-terminal extension generated a kinetic-folding intermediate that is not observed in the shorter construct. Kinetic experiments performed on a construct with a different amino acid sequence in the extension showed that the length and the sequence of the extension both contribute to the observed equilibrium and kinetic effects. These results point to an important role for the N terminus in the folding of FKBP and suggest that a biological consequence of N-terminal methionine removal observed in many eukaryotic and prokaryotic proteins is to increase the folding efficiency of the polypeptide chain.
Asunto(s)
Pliegue de Proteína , Proteínas de Unión a Tacrolimus/química , Cinética , Modelos Moleculares , Conformación Proteica , Desnaturalización Proteica , Renaturación de Proteína , Proteínas de Unión a Tacrolimus/metabolismo , TermodinámicaRESUMEN
The potential proteolysis sites of human TNF are considered. By site-directed mutagenesis the Arg-31 residue of mature TNF was substituted by Gln. The analysis of cytotoxicity of initial and mutant (R31Q) proteins on mouse L929 fibroblasts did not reveal any differences in biological activity. For the mutant protein a change in proteolysis dynamics was shown in contrast to the natural variant: mutant TNF displayed increased stability when treated with trypsin.
