Orthogonal protein purification--expanding the repertoire of GST fusion systems.
We have previously developed a labeling scheme that can be used to site-specifically link human glutathione transferases (hGSTs) from the alpha class to chemical entities such as fluorophores and aldehydes. The reagents are in-house synthesized derivatives of glutathione (GS-derivatives). We have focused on a lysine mutant of hGST A1:A216K. In this study, we wanted to utilize these findings and improve on protein purification schemes that are using GSTs as fusion partners. We have used random mutagenesis to scramble the hydrophobic binding site of A216K through mutations at position M208 and isolated a library of 11 A216K/M208X mutants. All mutants were easily expressed and purified and retained all or parts of the catalytic properties of the parent GST. The mutants were stable over several days at room temperature. The A216K/M208X mutants could be site-specifically labeled using our designed fluorescent reagents. Furthermore, reaction with an aldehyde-containing reagent termed GS-Al results in site-specific introduction of an orthogonal handle for subsequent conjugation with aldehyde-reactive probes. Labeling with coumarin results in a fluorescent protein-conjugate that can bind glutathione (GSH) derivatives for subsequent affinity purification. The K(d) for S-hexyl-GSH of coumarin-labeled A216K was measured to be 2.5 microM. The candidate proteins A216K and A216K/M208F could be purified in high yield in a one-step procedure through affinity chromatography (Glutathione Sepharose 4B). The proteins can readily be perceived as improved GST fusion partners.
Viljanen
Johan
J
IFM, Linköping University, S-581 83 Linköping, Sweden.
Larsson
Jenny
J
Broo
Kerstin S
KS
D000215:Acylation; D000447:Aldehydes / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002846:Chromatography, Affinity; D003062:Codon; D003374:Coumarins / Q000378:metabolism; D003460:Crystallization; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D004952:Esters / Q000737:chemistry; D005456:Fluorescent Dyes / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D008956:Models, Chemical; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010447:Peptide Hydrolases / Q000494:pharmacology; D019151:Peptide Library; D011485:Protein Binding; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D013997:Time Factors
D000447:Aldehydes; D003062:Codon; D003374:Coumarins; D004952:Esters; D005456:Fluorescent Dyes; D007527:Isoenzymes; D019151:Peptide Library; D011506:Proteins; D011993:Recombinant Fusion Proteins; C030123:coumarin; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha; D010447:Peptide Hydrolases; D008239:Lysine