Iavi jcsg tsri crystalmation robotic system

The Fab fragments of LMCA-CDRL3_SAR, PCT64-13C (early Fab), and PCT64-35S (late Fab) were expressed in FreeStyle 293F cells (Invitrogen), with a heavy chain:light chain ratio of 2:1 and co-transfection with protein-tyrosine sulfotransferase 1 (TPST1) to enhance tyrosine sulfation. Fab fragments were subsequently purified by affinity chromatography (anti-human kappa) and cation exchange chromatography (Mono S 10/100 GL). Purified fractions were analyzed by gel electrophoresis and exchanged into 20 mM sodium acetate (pH 5.6; LMCA-CDRL3_SAR) and 20 mM Tris and 150 mM NaCl (pH 7.4; early and late Fab), respectively. The proteins were subjected to crystallization trials with the IAVI/JCSG/TSRI CrystalMation robotic system (Rigaku) at either 20°C (LMCA-CDRL3_SAR) or both 4°C and 20°C (early and late Fab).

Fab fragments of the respective Abs (PC39 UCA, 17A, 23D, 50C, 50E, 50I, 50L and 55C) were expressed in FreeStyle 293F cells (Invitrogen), at a 2:1 ratio of heavy to light chain and harvested 5–6 days after transfection. Fragments were purified by affinity chromatography (anti-human kappa—GE) and cation exchange chromatography (Mono S 10/100 GL), as previously described [104 (link)]. Purified fractions were analyzed by gel electrophoresis, concentrated, and subjected to crystallization trials with the IAVI/JCSG/TSRI CrystalMation robotic system (Rigaku) at 20°C, either in the cation exchange elution buffer, or after exchange into 20 mM Tris, 150 mM NaCl, pH 7.4 (Fab 17A).

VRC01 Fab was produced and purified as previously described [40 (link)]. eOD-N276Kif, a minimal glycan, alanine-resurfaced construct possessing only three glycosylation sites (N18, N65 and N79, eOD numbering as in PDB IDs 4JPJ and 4JPK) was designed and subsequently transfected in HEK 293F (Invitrogen) suspension cells in the presence of the mannosidase I inhibitor kifunensine, to yield homogeneous Man₉GlcNAc₂ carbohydrates. The secreted protein was purified via its C-terminus His₆-tag by affinity chromatography using HisTrap nickel columns (GE Healthcare), and subsequently purified to size homogeneity by Superdex 200 gel filtration chromatography (GE Healthcare). After incubation of VRC01 Fab in molar excess of eOD-N276Kif, the complex was purified by Superdex 200 size exclusion chromatography (GE Healthcare) and concentrated to ~5 mg/ml for crystallization trials using the automated JCSG/IAVI/TSRI CrystalMation robotic system (Rigaku) at the Joint Center for Structural Genomics (www.jcsg.org) at TSRI. Crystals used for data collection were obtained with 0.16 M ammonium sulfate, 20% (w/v) PEG4000, 20% (v/v) glycerol, 0.08 M sodium acetate, pH 4.6, as the mother liquor. Prior to data collection, crystals were cryoprotected in the mother liquor supplemented with 40% glycerol and subsequently fast-plunged into liquid nitrogen.