Purification of recombinant adenomatous polyposis coli polypeptide chains from E. coli extracts by continuous-elution electrophoresis.
A polypeptide chain encoded by the exons 1-3 of the human adenomatous polyposis coli (APC) tumor suppressor gene was expressed as a maltose binding fusion protein (MBP) in E. coli to be used for immunization purposes. It turned out, that the APC-MBP fusion product of 60 kDa was deposited in bacterial inclusion bodies and, in addition, it was not retarded by an amylose affinity column, most probably due to an altered conformation of the chimeric molecule. For this reason, we established an alternative purification scheme, which took advantage of SDS-extraction followed by a high-resolution two-step continuous-elution electrophoresis (CEE) procedure. This purification method allowed us to obtain high yields of pure human APC exon 1-3-encoded proteins. The final yield of the pure APC polypeptide chains was estimated to represent 5-8% of the amount of SDS-extracted E. coli lysate subjected to the first cycle of CEE. The purified APC molecules were successfully used for the development of specific antibodies. The CEE procedure described here represents a general purification method which is valuable in cases where fusion proteins are deposited as inclusion bodies in bacteria, or if affinity chromatography is precluded due to a conformation-induced lack of ligand binding of the chimeric molecule.
Kraus
C
C
Institut fuer Humangenetik, Universitaet Erlangen-Nuernberg, Germany.
Klein
E
E
Ballhausen
W G
WG
D011125:Adenomatous Polyposis Coli / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004586:Electrophoresis / Q000379:methods; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D010452:Peptide Biosynthesis; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D009363:Neoplasm Proteins; D010455:Peptides; D011994:Recombinant Proteins