Abstract
We have constructed a human V(H) library based on a camelized V(H) sequence. The library was constructed with complete randomization of 19 of the 23 CDR3 residues and was panned against two monoclonal antibody targets to generate V(H) sequences for determination of the antigen contact residue positions. Furthermore, the feasibility and desirability of introducing a disulfide bridge between CDR1 and CDR3 was investigated. Sequences derived from the library showed a bias toward the use of C-terminal CDR3 residues as antigen contact residues. Mass spectrometric analyses indicated that CDR1-CDR3 disulfide formation was universal. However, surface plasmon resonance and NMR data showed that the CDR3 constraint imposed by the disulfide bridge was not always desirable. Very high yields of soluble protein products and lack of protein aggregation, as demonstrated by the quality of the (1)H-(15)N HSQC spectra, indicated that the V(H) sequence for library construction was a good choice. These results should be useful in the design of V(H) libraries with optimal features.
Highlights
Heavy chain antibodies, found in camelids [1, 2], lack light chains and as a result have variable domains that reflect the absence of a VL partner
Camelization of human VHs is a promising technology for the generation of small antigen-binding fragments that should be useful for therapeutic purposes in humans
The yields in E. coli of soluble camelized product were low and in order to obtain the yields and stability required for NMR studies they opted for a W47I mutation instead of the W47G mutation [5, 13]
Summary
Library Construction and Panning—Wild type dAb (Fig. 1) was constructed from BT32/A6 [18]. To sub-clone the library into a phage vector, library phagemid DNA template (180 pmol) and two primers which were complimentary to the 5Ј and 3Ј ends of the dAb genes, and incorporated flanking ApaLI and NotI restriction sites, were used to PCR amplify the dAb genes. Serial dilutions of infected cells were used to determine the titers of eluted phage as described above. The remainders of the infected cells were pelleted, re-suspended in 900 l of 2 ϫ YT [19], mixed in 300-l aliquots with 3 ml of 0.7% agarose in LB [19] at 50 °C and the phage propagated on plates overnight at 37 °C. A 25 M excess of DTT, relative to Cys residues, was added and the mixture was incubated at room temperature for 30 min.
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