title: Addition of a polypeptide stretch at the N-terminus improves the expression, stability and solubility of recombinant protein tyrosine phosphatases from Drosophila melanogaster.
abstract: The production of recombinant proteins in Escherichia coli involves substantial optimization in the size of the protein and over-expression strategies to avoid inclusion-body formation. Here we report our observations on this so-called construct dependence using the catalytic domains of five Drosophila melanogaster receptor protein tyrosine phosphatases as a model system. Five strains of E. coli as well as three variations in purification tags viz., poly-histidine peptide attachments at the N- and C-termini and a construct with Glutathione-S-transferase at the N-terminus were examined. In this study we observe that inclusion of a 45 residue stretch at the N-terminus was crucial for over-expression of the enzymes, influencing both the solubility and the stability of these recombinant proteins. While the addition of negatively charged residues in the N-terminal extension could partially rationalize the improvement in the solubility of these constructs, conventional parameters like the proportion of order promoting residues or aliphatic index did not correlate with the improved biochemical characteristics. These findings thus suggest the inclusion of additional parameters apart from rigid domain predictions to obtain domain constructs that are most likely to yield soluble protein upon expression in E. coli.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: Lalima L
authors.initials: LL
authors.lastname: Madan
authors.forename: B
authors.initials: B
authors.lastname: Gopal
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D004331:Drosophila melanogaster / Q000201:enzymology*; D004795:Enzyme Stability; D004926:Escherichia coli; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism*; D012995:Solubility; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; D017027:Protein Tyrosine Phosphatases