Document Detail


Functional expression in Pichia pastoris of human and rat intrinsic factor.
MedLine Citation:
PMID:  10786616     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
J Wen; M B Kinnear; M A Richardson; N S Willetts; G J Russell-Jones; M M Gordon; D H Alpers
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biochimica et biophysica acta     Volume:  1490     ISSN:  0006-3002     ISO Abbreviation:  Biochim. Biophys. Acta     Publication Date:  2000 Jan 
Date Detail:
Created Date:  2000-05-24     Completed Date:  2000-05-24     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0217513     Medline TA:  Biochim Biophys Acta     Country:  NETHERLANDS    
Other Details:
Languages:  eng     Pagination:  43-53     Citation Subset:  IM    
Affiliation:
Division of Gastroenterology, Washington University School of Medicine, St. Louis, MO 63110, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Cell Line
Genetic Vectors
Humans
Intrinsic Factor / biosynthesis*,  chemistry,  genetics
Kidney / metabolism
Microvilli / metabolism
Mutagenesis, Site-Directed
Pichia / genetics,  metabolism*
Rats
Receptors, Cell Surface / chemistry,  metabolism
Recombinant Proteins / biosynthesis
Grant Support
ID/Acronym/Agency:
P01 DK33487/DK/NIDDK NIH HHS
Chemical
Reg. No./Substance:
0/Receptors, Cell Surface; 0/Recombinant Proteins; 0/intrinsic factor-cobalamin receptor; 9008-12-2/Intrinsic Factor

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