Crystallization and preliminary crystallographic study of an Fab fragment of a pathogenic rat monoclonal antibody against the nicotinic acetylcholine receptor

Crystal structure of Fab198, an efficient protector of the acetylcholine receptor against myasthenogenic antibodies

“…Under nonreducing conditions all Fab aliquots migrated as single bands at approximately 50 kDa, while under reducing conditions they migrated as two adjacent bands at 25 kDa. However, analysis by analytical isoelectric focusing using agarose gel (Pharmacia, Sweden), revealed heterogeneity in pI, well described earlier [21–23], which is known to prevent the growth of diffraction‐quality crystals. The high frequency of Asn residues in the l chain (see below) may explain the pI heterogeneity observed.…”

“…Crystallization conditions were screened taking into account the successful trials, described by Vatzaki et al . [21]. Crystallization trials were carried out using the hanging and sitting drop techniques at 18 °C at a protein concentration of 12 mg·mL −1 .…”

“…Here we present the structural analysis at 2.8 Å resolution of the Fab fragment of the anti‐MIR mAb198. Crystallization conditions and preliminary crystallographic data on Fab198 have previously been reported [21]. However, in order to obtain better quality crystals, new purification methods and crystallization conditions were followed.…”

Characterisation, crystallisation and preliminary X‐ray diffraction analysis of a Fab fragment of a rat monoclonal antibody with very high affinity for the human muscle acetylcholine receptor

“…However, when the same fractions were analysed by analytical isoelectric focusing (IEF) using agarose gel (Pharmacia), each of the two Fab containing peaks was analysed into several bands. This heterogeneity in p1 is observed in several cases of Fabs [15,16] and is known to prevent the growth of diffraction quality crystals. Therefore, further purification with the chromatofocusing column was an essential step.…”