Functional expression in Pichia pastoris of human and rat intrinsic factor.
Intrinsic factor (IF) has been expressed previously in Baculovirus with a yield (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 chromatography. Expression of recombinant proteins revealed a major band of 49 kDa for both human and rat IF. Expression of human IF was achieved at 1040 mg/l, but of rat IF at only 1-2 mg/l. Reaction of human IF with a photo-activatable derivative of Cbl was demonstrated by Western blotting, and detection of IF fragments by anti-Cbl monoclonal antibody and by amino-terminal sequencing revealed at least three regions (residues 129-151, 234-254, and +294) linked to Cbl. Both recombinant human and rat [125I]IF-Cbl bound to rat and guinea pig brush border membranes with similar affinity, but the binding capacity 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 region 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 26/27 (Glu26 to Asp and Asn27 to Gln) and 32/34 (Ser32 to Thr and Tyr34 to Arg) showed changes in both Ka and Vmax, with great effects on Vmax. In conclusion, P. pastoris is an expression system that produces functional human IF at a higher yield than in the baculovirus system. Cbl binding was directly demonstrated at multiple sites along the linear sequence of human IF. The receptor binding function of the amino terminal sequence 25 62 has been confirmed, but it is insufficient to reproduce all the features of IF-Cbl binding.
Wen
J
J
Division of Gastroenterology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Kinnear
M B
MB
Richardson
M A
MA
Willetts
N S
NS
Russell-Jones
G J
GJ
Gordon
M M
MM
Alpers
D H
DH
D000818:Animals; D002460:Cell Line; D005822:Genetic Vectors; D006801:Humans; D007437:Intrinsic Factor / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D007668:Kidney / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis
D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C029486:intrinsic factor-cobalamin receptor; D007437:Intrinsic Factor