Low temperature pepsin proteolysis: An effective procedure for mouse IgM F(ab′) 2 fragment production

Abstract

Proteolytic fragments from murine IgM antibodies can be obtained by a variety of enzymatic procedures including pepsin digestion. However, these procedures exhibit considerable variability with respect to the size and yield of fragments. In this report, we describe a single step enzymatic digestion procedure which, when performed at a low temperature, can generate reasonably homogeneous proteolytic fragments with > 90% yield. Murine IgM monoclonal antibodies specific for the DNP moiety were used throughout this study. By subjecting intact IgM antibodies to enzymatic digestion with pepsin under mildly acidic conditions at 4°C, covalent and noncovalent F(ab′) 2 fragments were generated. Under nonreducing conditions, the covalent F(ab′) 2 fragments migrated with an apparent molecular weight of 134 kDa on SDS-PAGE, while the noncovalent F(ab′) 2 fragments dissociated into their respective Fab′ fragments each exhibiting an apparent molecular weight of 67 kDa. However, when subjected to gel-filtration chromatography in nondenaturing buffers, each of these F(ab′) 2 fragments eluted in a volume corresponding to 130–140 kDa. Upon extensive reduction, these fragments demonstrated equimolar concentrations of shortened μ chains, termed F d′ fragments, and light chains. The binding activity of these F(ab′) 2 fragments was unaffected by the digestion. The F(ab′) 2 fragments exhibited the same number of binding sites and binding affinities as their respective homologous reductive subunits when analysed by equilibrium dialysis. To test the efficacy of this procedure, peptic digestions were also performed at 37°C. Considerably lower yields of F(ab′) 2 fragments were obtained when compared to digestion at 4°C.