Resolution of proteins on a phenyl-Superose HR5/5 column and its application to examining the conformation homogeneity of refolded recombinant staphylococcal nuclease.
In order to examine the effect of amino acid substitutions on protein retention in hydrophobic interaction chromatography and the resolution of a phenyl-Superose HR5/5 column, two groups of staphylococcal nucleases, named Y113/W140 (wild-type), Y113W/W140 and Y113/W140F, Y113W/W140F, were produced by substituting tryptophan (W) for tyrosine (Y) at residue 113 and phenylalanine (F) for tryptophan (W) at residue 140. For each group, the proteins have the same amino acid at residue 140, but a different amino acid at residue 113. The solvent perturbation of nuclease fluorescence and 1,8-anilinoaphthalene-8-sulfonate binding studies showed that the substitutions do not change the side-chain positions of amino acids at residues 113 and 140. Chromatography of the proteins on the Phenyl-Superose HR5/5 column showed that the proteins with tryptophan at residue 113 have longer retention times than the proteins having tyrosine at residue 113; the proteins with the same amino acid at residue 113 have almost the same retention time regardless of substituting phenylalanine for tryptophan at residue 140. The studies clearly indicate that not all amino acid substitutions have an effect on protein retention; the contribution to retention of a given amino acid substitution depends on its position in a protein. Single amino acid substitutions at the exterior surface of a protein, which change the strength of hydrophobic interaction, can affect the protein retention in hydrophobic interaction chromatography. Staphylococcal nuclease and its mutants with only one amino acid difference on their surfaces can be discriminated by the phenyl-Superose column.(ABSTRACT TRUNCATED AT 250 WORDS)
Institute of Biophysics, Academia Sinica, Beijing, China.
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G
Jing
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Zhou
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Liu
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Zhou
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Liu
D000596:Amino Acids / Q000737:chemistry; D002850:Chromatography, Gel; D002942:Circular Dichroism; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000302:isolation & purification*; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012685:Sepharose / Q000031:analogs & derivatives*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D011994:Recombinant Proteins; C020927:phenyl-sepharose; D012685:Sepharose; D008836:Micrococcal Nuclease