Functional expression in Pichia pastoris of human and rat intrinsic factor

Intrinsic factor (IF) has been expressed previously in Baculovirus with a y ield (0.1-1 mg/l) that was inadequate for structural and metabolic studies. IF cDNAs were cloned into the shuttle vector pPIC9 of P. pastoris, and the proteins were induced and purified by cobalamin (Cbl) affinity chromatogra phy. Expression of recombinant proteins revealed a major band of 49 kDa for both human and rat IF. Expression of human IF was achieved at 10-40 mg/l, but of rat IF at only 1-2 mg/l. Reaction of human IF with a photo-activatab le derivative of Cbl was demonstrated by Western blotting, and detection of IF fragments by anti-Cbl monoclonal antibody and by amino-terminal sequenc ing revealed at least three regions (residues 129-151, 234-254, and +294) l inked to Cbl. Both recombinant human and rat [I-125]IF-Cbl bound to rat and guinea pig brush border membranes with similar affinity, but the binding c apacity of human IF for the rat receptor was only 10% compared with rat IF. All six amino acids within the previously identified N-terminal binding re gion of human IF were mutated to be identical to rat IF, but the resulting chimeric IF still bound poorly to rat membranes. Mutations of residues 26/2 7 (Glu(26) to Asp and Asn(27) to Gln) and 32/34 (Ser(32) to Thr and Tyr(34) to Arg) showed changes in both K-a and V-max, with great effects on V-max. In conclusion, P. pastoris is an expression system that produces functiona l human IF at a higher yield than in the baculovirus system. Cbl binding wa s directly demonstrated at multiple sites along the linear sequence of huma n IF. The receptor binding function of the amino terminal sequence 25-62 ha s been confirmed, but it is insufficient to reproduce all the features of I F-Cbl binding. (C) 2000 Elsevier Science B.V. All rights reserved.