title: Increased biological activity of a recombinant factor IX variant carrying alanine at position +1.
abstract: In attempts to improve the post-translational modification and processing of recombinant factor IX (FIX) we have altered the cDNA sequence encoding pre-pro-FIX using site-directed mutagenesis and have expressed the variant cDNAs in BHK21 cells using a vaccinia-virus-derived vector. We find that substitution of the tyrosine residue at +1 for an alanine increases the biological activity of the recombinant molecules 2-fold. On the other hand, substitution of the proline at -3 for a valine results in no significant change to the specific activity of the protein. Other alterations to the N-terminus of the FIX proteins, in attempts to mimic other vitamin-K-dependent proteins, result in the failure to produce a secreted polypeptide. N-terminal sequence analysis of purified recombinant molecules reveals a correlation between specific activity and the efficiency of correct pro-sequence cleavage. gamma-Carboxylation analysis of purified recombinant proteins indicates that each molecule including unmutated FIX is completely gamma-carboxylated in this system. Thus the observed increase in biological activity of FIX variants containing an alanine at position +1 is not due to increased gamma-carboxylation but, at least in part, to more efficient pro-peptide cleavage.
authors.affiliation: Transgène S.A., Strasbourg, France.
authors.forename: P
authors.initials: P
authors.lastname: Meulien
authors.forename: A
authors.initials: A
authors.lastname: Balland
authors.forename: P
authors.initials: P
authors.lastname: Lepage
authors.forename: F
authors.initials: F
authors.lastname: Mischler
authors.forename: K
authors.initials: K
authors.lastname: Dott
authors.forename: C
authors.initials: C
authors.lastname: Hauss
authors.forename: M
authors.initials: M
authors.lastname: Grandgeorge
authors.forename: J P
authors.initials: JP
authors.lastname: Lecocq
mesh_terms: D000409:Alanine / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002872:Chromosome Deletion; D006224:Cricetinae; D004247:DNA / Q000032:analysis; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D014644:Genetic Variation; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D014443:Tyrosine / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D014443:Tyrosine; D005164:Factor IX; D004247:DNA; D000409:Alanine