Expression, purification, and characterization of rat protein tyrosine phosphatase eta catalytic domain.
Receptor-like protein tyrosine phosphatases generally contain one or two conserved intracellular catalytic domains with a conserved sequence motif ([I/V]HCXAGXXR[S/T]G), a single transmembrane domain, and an external highly variable part. Here, we describe cloning of the intracellular catalytic domain of the rat protein tyrosine phosphatase eta (rPTPetaCD) into pET28a(+) vector, its expression in Escherichia coli, purification and initial characterization. The purification of His6-tagged rPTPetaCD to near homogeneity was achieved by a combination of affinity and size exclusion chromatography. The His-tag was subsequently removed by thrombin digestion. PhastGel IEF electrophoresis demonstrated that the isoelectric point of this 41 kDa His6-tag free recombinant protein was 7.3, which is just slightly higher than the theoretically predicted value of 7.2. To assess the functionality of the rPTPetaCD we used the pNPP hydrolysis assay and observed that the enzyme has a specific activity of 9 nmol/min/mug. The secondary structure and stability of the recombinant protein was also analyzed by circular dichroism and fluorescence spectroscopy. In summary, the rPTPetaCD is stable at 18 degrees C, properly folded, and fully active, which makes it a suitable candidate for structural and functional studies.
Santos
Maria A M
MA
Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, Avenida Trabalhador São Carlense, 400, CEP 13566-590 São Carlos, SP, Brazil.
Santos
Silvia M
SM
Matozo
Huita C
HC
Portugal
Rodrigo V
RV
Iuliano
Rodolfo
R
Fusco
Alfredo
A
Polikarpov
Igor
I
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
D011994:Recombinant Proteins; D017027:Protein Tyrosine Phosphatases; C453859:phosphotyrosine phosphatase eta