EFFICIENT SECRETION OF MURINE FAB FRAGMENTS BY ESCHERICHIA-COLI IS DETERMINED BY THE 1ST CONSTANT DOMAIN OF THE HEAVY-CHAIN

Fab fragments of IgG1 and IgG3 subclass antibodies which bind to 2-phe nyloxazolone (Ox) were produced in Escherichia coli. The signal sequen ces of the Fd and L chains were correctly processed, the fragments wer e secreted into the periplasmic space and released into the culture me dium upon prolonged cultivations. The yields of active Ox IgG1 and Ox IgG3 Fab fragments after one-step purification from the culture medium by affinity chromatography were 2 mug/ml and 0.5 mug/ml, respectively . The majority of the purified Ox IgG1 Fab was properly assembled, but in the case of Ox IgG3, the preparation was found to consist of a com plete L chain and C-terminally degraded fragments of the Fd chain. A d eletion up to the interchain disulfide bond in the first constant doma in (CH1) of the Ox IgG3 Fd chain led to proper assembly of the truncat ed Fab fragment. The production level of the truncated fragment was co mparable to that of the Ox IgG1 Fab and its hapten-binding activity si milar to that of the idiotype monoclonal antibody. The temperature sta bility of the Ox IgG1 Fab was similar to that of the intact antibody. However, both of the Ox IgG3 Fab fragments showed reduced stability, s uggesting that the CH1 domain contributes significantly to the thermal stability of the Fab fragment.