title: N-terminal tagged lactate dehydrogenase proteins: evaluation of relative hydrophobicity by hydrophobic interaction chromatography and aqueous two-phase system partition.
abstract: The hydrophobic contributions of 17 individual peptides, fused to the N-terminal of Bacillus stearothermophilus lactate dehydrogenase (LDH) were studied by hydrophobic interaction chromatography (HIC) and aqueous two-phase system (ATPS). The constructs were sequenced from a protein library designed with a five-amino acid randomised region in the N-terminal of an LDH protein. The 17 LDH variants and an LDH control lacking the randomised region were expressed in Escherichia coli. HIC and ATPS behaviour of the proteins indicated significant differences in protein hydrophobicity, even though the modifications caused only 1% increase in protein molecular weight and 2% variation in isoelectric points. HIC and ATPS results correlated well (R(2) = 0.89). Protein expression was clearly affected by N-terminal modification, but there was no evidence that the modification affected protein activity. A GluAsnAlaAspVal modification resulted in increased protein expression. In most cases, HIC and ATPS results compared favourably with those predicted on the basis of 34 amino acid residue hydrophobicity scales; assuming exposure of tag residues to solution. Exceptions included LeuAlaGlyValIle and LeuTyrGlyCysIle modifications, which were predicted, assuming full solution exposure, to be more hydrophobic than observed.
authors.affiliation: Department of Pure and Applied Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, PO Box 124, SE-221 00 Lund, Sweden.
authors.forename: Sara
authors.initials: S
authors.lastname: Fexby
authors.forename: Henrik
authors.initials: H
authors.lastname: Ihre
authors.forename: James
authors.initials: J
authors.lastname: Van Alstine
authors.forename: Leif
authors.initials: L
authors.lastname: Bülow
mesh_terms: D001483:Base Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D017931:DNA Primers; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D007770:L-Lactate Dehydrogenase