Expression of mouse uterine peptidylarginine deiminase in Escherichia coli: construction of expression plasmid and properties of the recombinant enzyme.
To study the structure/function relationships of peptidylarginine deiminase (PAD), we constructed an Escherichia coli expression plasmid for mouse uterine PAD. First, segments of a cDNA encoding murine PAD were subcloned into a single plasmid, and the resulting plasmid, pKSPAD1, was inserted into an expression vector, pKK223-3, at the EcoRI and HindIII restriction sites. Since no detectable amount or activity of the PAD was produced by E. coli carrying that plasmid, the 5'-untranslated sequence of the cDNA was replaced with several synthetic DNAs. One of the constructed plasmids, pKKPAD4, which had a unique DNA linker containing a pair of Shine-Dalgarno sequences and a short preceding cistron inserted into the adjacent 5'-region of the coding region, produced a large quantity of mouse PAD as an unfused protein in E. coli. The purified recombinant PAD was indistinguishable from the native enzyme with respect to some structural properties, such as molecular mass, amino- and carboxyl-terminal sequences, and circular dichroism spectra. However, the alpha-amino group of the amino-terminal methionine residue of the recombinant PAD was not acetylated as was that of the native enzyme. Comparison of the recombinant PAD with the natural enzyme did not indicate significant differences in their sensitivity to activation by Ca2+ and in their substrate specificity toward arginine derivatives. The rates of modification of soybean trypsin inhibitor (Kunitz) were also similar for the recombinant and native PADs. These results indicate that the recombinant PAD has biological activities identical to those of the native enzyme and that the N alpha-acetyl group in the native PAD does not appear to have any particular role in the enzyme's catalytic function.
Ohsugi
I
I
Laboratory of Biochemistry, School of Agriculture, Ibaraki University, Japan.
Takahara
H
H
Shiraiwa
M
M
Sugawara
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000652:urine; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010957:Plasmids / Q000235:genetics; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D011994:Recombinant Proteins; D006867:Hydrolases; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; C552151:peptidylarginine deiminase 4, mouse