Structure-based stability engineering of the mouse IgG1 Fab fragment by modifying constant domains.
A semi-rational approach based on structural data was exploited in a search for CH1 and CL domains with improved intrinsic thermodynamic stabilities. Structural and amino acid level comparisons were carried out against known biophysically well-behaving and thermodynamically beneficial scFv and Fab fragments. A number of mutant Fab fragments were constructed by site-directed mutagenesis of regions in the CH1 and CL domains expected to be most sensitive under physical stress conditions. These mutations were located on three sites in the Fab constant domains; a mobile loop in the CH1 domain, residues surrounding the two largest solvated hydrophobic cavities located in the interface of the CH1 and CL domains and the hydrophobic core regions of both CH1 and CL. Expression levels of functional Fab fragments, denaturant-induced unfolding equilibria and circular dichroism spectroscopy were used to evaluate the relative stabilities of the wild-type and the mutant Fab fragments. The highest thermodynamic stability was reached through the mutation strategy, where the hydrophobicity and the packing density of the solvated hydrophobic cavity in the CH1/CL interface was increased by the replacement of the hydrophilic Thr178 in the CL domain by a more hydrophobic residue, valine or isoleucine. The midpoint of the transition curve from native to unfolded states of the protein, measured by fluorescence emission, occurred at concentrations of guanidine hydrochloride of 2.4 M and 2.6 M for the wild-type Fab and the most stable mutants, respectively. Our results illustrate that point mutations targeted to the CH1/CL interface were advantageous for the overall thermodynamic stability of the Fab fragment.
Teerinen
Tuija
T
VTT Biotechnology, P.O. Box 1000, 02044 VTT Espoo, Finland.
Valjakka
Jarkko
J
Rouvinen
Juha
J
Takkinen
Kristiina
K
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D007127:Immunoglobulin Constant Regions / Q000737:chemistry* / Q000235:genetics; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D013739:Testosterone / Q000276:immunology; D013816:Thermodynamics
D007127:Immunoglobulin Constant Regions; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D013739:Testosterone