Optimization of the crystallizability of a single-chain antibody fragment

Abstract

Single-chain variable antibody fragments (scFvs) are molecules with immense therapeutic and diagnostic potential. Knowledge of their three-dimensional structure is important for understanding their antigen-binding mode as well as for protein-engineering approaches such as antibody humanization. A major obstacle to the crystallization of single-chain variable antibody fragments is their relatively poor homogeneity caused by spontaneous oligomerization. A new approach to optimization of the crystallizability of single-chain variable antibody fragments is demonstrated using a representative single-chain variable fragment derived from the anti-CD3 antibody MEM-57. A Thermofluor-based assay was utilized to screen for optimal conditions for antibody-fragment stability and homogeneity. Such an optimization of the protein storage buffer led to a significantly improved ability of the scFv MEM-57 to yield crystals.

Keywords: Thermofluor assay; crystallizability optimization; crystallization; differential scanning fluorimetry; oligomerization; single-chain antibody fragment.

Figure 1

Scheme of the scFv MEM-57 antibody fragment. (a) A scheme of the scFv MEM-57 gene construct and the corresponding protein product; V_H and V_L are the variable domains of the heavy and light immunoglobulin chains, respectively, pelB is a leader sequence targeting recombinant product to the periplasm, c-Myc is a ten-amino-acid epitope and His₅ is the pentahistidine purification tag. (b) Quaternary structure of the scFv MEM-57 protein. In addition to scFv monomers, domain-swapped inter-chain scFv dimers can be formed. (c) The purity of scFv MEM-57 is shown in lane 1 on a silver-stained denaturing SDS–PAGE gel under nonreducing conditions; the molecular-weight standard is in lane 3 (molecular weights are indicated in kDa) and lane 2 is empty.

Figure 2

Inhomogeneity of scFv MEM-57 in the original storage buffer (20 mM diethanolamine pH 8.4, 100 mM NaCl). (a) Size-exclusion chromatographic analysis of scFv MEM-57 at 7.5 mg ml⁻¹. Apparent molecular weights are indicated for each peak together with the percentage of the individual oligomeric forms in the total protein content. (b) The DLS particle-size distribution reveals polydispersity of scFv MEM-57 at 15 mg ml⁻¹.

Figure 3

Thermofluor assay for scFv MEM-57. (a) Thermofluor-based protein-unfolding curves for the original (black) and optimized (green) storage buffers are shown along with representative multiple-inflection curves which were excluded from further melting-temperature (T _m) analysis (blue). (b) Melting temperatures for storage buffers with the melting temperatures of other single-inflection buffers. The original and optimized storage buffers are highlighted in black and green, respectively.

Figure 4

Homogeneity of scFv MEM-57 in the optimized storage buffer (100 mM sodium phosphate pH 7.5, 200 mM NaCl). (a) Size-exclusion chromatographic analysis of scFv MEM-57 at 7.5 mg ml⁻¹. Apparent molecular weights are indicated for each peak together with the percentage of the individual oligomeric forms in the total protein content. The discrepancy in the apparent molecular weights for the scFv dimer in the original and optimized buffer (compare with Fig. 2 ▶ a) might be caused by the regeneration and recalibration of the SEC column between these two runs. (b) The DLS particle-size distribution reveals monodispersity of scFv MEM-57 at 15 mg ml⁻¹.

Figure 5

Crystallization of scFv MEM-57 in the optimized storage buffer. (a) A scheme of the crystallization screening plate containing The PEGs Suite (Qiagen, Netherlands) is shown with the conditions yielding crystals highlighted in green. (b) An example of crystals obtained using 100 mM HEPES pH 7.5, 15%(w/v) PEG 20 000. (c) An example of crystals obtained using 200 mM sodium fluoride, 20%(w/v) PEG 3350. (d) An example of crystals obtained using 100 mM HEPES pH 7.5, 25%(w/v) PEG 3000.