Hydrophobicity engineering of cholera toxin A1 subunit in the strong adjuvant fusion protein CTA1-DD.
Protein engineering of the cholera toxin A1 subunit (CTA1) fused to a dimer of the Ig-binding D-region of Staphylococcus aureus protein A (DD) was employed to investigate the effect of specific amino acid changes on solubility, stability, enzymatic activity and capacity to act as an adjuvant in vivo. A series of CTA1-DD analogues were selected by a rational modeling approach, in which surface-exposed hydrophobic amino acids of CTA1 were exchanged for hydrophilic counterparts modeled for best structural fit. Of six different mutants initially produced, two analogues, CTA1Phe132Ser-DD and CTA1Pro185Gln-DD, were demonstrated to have 50 and 70% increased solubility, respectively, at neutral pH. The double mutant CTA1Phe132Ser/Pro185Gln-DD was at least threefold more soluble, demonstrating an additive effect of the two mutations. Only the Phe132Ser analogue retained full biological activity and stability compared with the native CTA1-DD fusion protein. Two mutants, Pro185Gln and Phe31His mutations, exhibited unaltered ADP-ribosyltransferase activity in vitro, but demonstrated markedly reduced adjuvant function. Since the Pro185 and Phe31 amino acids are located in close vicinity on the distal side of the molecule relative to the enzymatically active cleft, it is conceivable that this region is involved in mediating a biological function, separate from the enzymatic activity but intrinsic to the adjuvant activity of CTA1.
Agren
L
L
Department of Medical Microbiology and Immunology, University of Göteborg, Sweden.
Norin
M
M
Lycke
N
N
Löwenadler
B
B
D000818:Animals; D002772:Cholera Toxin / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D008957:Models, Genetic; D008958:Models, Molecular; D016296:Mutagenesis; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins; D012995:Solubility; D013205:Staphylococcal Protein A / Q000737:chemistry; D013211:Staphylococcus aureus / Q000737:chemistry
D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D002772:Cholera Toxin; D011065:Poly(ADP-ribose) Polymerases