Abstract
We report a comprehensive analysis of sequence features that allow for the production of autonomous human heavy chain variable (V(H)) domains that are stable and soluble in the absence of a light chain partner. Using combinatorial phage-displayed libraries and conventional biophysical methods, we analyzed the entire former light chain interface and the third complementarity determining region (CDR3). Unlike the monomeric variable domains of camelid heavy chain antibodies (V(H)H domains), in which autonomous behavior depends on interactions between the hydrophobic former light chain interface and CDR3, we find that the stability of many in vitro evolved V(H) domains is essentially independent of the CDR3 sequence and instead derives from mutations that increase the hydrophilicity of the former light chain interface by replacing exposed hydrophobic residues with structurally compatible hydrophilic substitutions. The engineered domains can be expressed recombinantly at high yield, are predominantly monomeric at high concentrations, unfold reversibly, and are even more thermostable than typical camelid V(H)H domains. Many of the stabilizing mutations are rare in natural V(H) and V(H)H domains and thus could not be predicted by studying natural sequences and structures. The results demonstrate that autonomous V(H) domains with structural properties beyond the scope of natural frameworks can be derived by using non-natural mutations, which differ from those found in camelid V(H)H domains. These findings should enable the development of libraries of synthetic V(H) domains with CDR3 diversities unconstrained by structural demands.
Highlights
EXPERIMENTAL PROCEDURESLibrary Construction and Analysis—For the phage display of VH-4D5, a phagemid (pPAB43778) was constructed from a previously described phagemid (pS1602) [43] by inserting a DNA fragment encoding VH-4D5 in place of the region encoding human growth hormone
Exceptions [2, 3]
There are a number of hallmark sequence changes in VHH domains relative to conventional VH domains, which function to reduce the hydrophobicity of the former light chain interface
Summary
Library Construction and Analysis—For the phage display of VH-4D5, a phagemid (pPAB43778) was constructed from a previously described phagemid (pS1602) [43] by inserting a DNA fragment encoding VH-4D5 in place of the region encoding human growth hormone. Quantitative saturation scanning, and the analysis of library B, the sequences were analyzed with the program SGCOUNT as described (46 – 48). Ϳ200 clones were sequenced from each library, and the data were normalized for codon bias in the NNK degenerate codon (e.g. the NNK codon contains three unique codons for Arg, and the occurrence of Arg was divided by 3). The fraction folded (␣) was calculated using the following equathe ␣ value for the sample cooled down to 25 °C after heating to a temperature that induced complete unfolding. Crystallization, Structure Determination, and Refinement—For crystallization, VH-B1a was purified by protein A-Sepharose chromatography, as described above, and the eluted protein (10 mg) was loaded on a SuperdexTM HiLoadTM 16/60 gel filtration column The coordinates and structure factors for the VH-B1a structure have been deposited in the RCSB Protein Data Bank (PDB code 3B9V)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
