Expression of human factor IX and its subfragments in Escherichia coli and generation of antibodies to the subfragments.
A cDNA clone encoding the entire human blood clotting factor IX (amino acids -3 to 415 has been placed under control of transcription and translation signals from bacteriophage T7 and expressed in Escherichia coli. The full-length cDNA and 13 different subfragments (which together cover the entire coding sequence of mature factor IX plus amino acids -40 to -19 of the prepro leader sequence) have each been joined to the coding sequence for the major capsid protein of T7 after the 326th codon and expressed as fusion proteins. All of the fusion proteins were insoluble, which facilitated their purification. A goat polyclonal antiserum against human factor IX reacted to different extents with the different fusion proteins, and rabbit polyclonal antibodies raised against the purified fusion proteins recognize the factor IX molecule, as demonstrated by immunoblotting techniques. Antibodies against at least one of the fusion proteins can also inhibit the biological activity of purified factor IX in a one-stage partial thromboplastin time bioassay. We expect these fusion proteins and the antibodies against them to be useful in studying the structure and function of factor IX.
Lin
S W
SW
Department of Biology, University of North Carolina, Chapel Hill 27514.
Dunn
J J
JJ
Studier
F W
FW
Stafford
D W
DW
D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics*; D005164:Factor IX / Q000235:genetics*; D006801:Humans; D010446:Peptide Fragments / Q000235:genetics; D010957:Plasmids; D014176:Protein Biosynthesis; D013604:T-Phages / Q000235:genetics; D014158:Transcription, Genetic
D010446:Peptide Fragments; D005164:Factor IX; D004247:DNA; D004262:DNA Restriction Enzymes