Purification of recombinant HBc antigen expressed in Escherichia coli and Pichia pastoris: comparison of size-exclusion chromatography and ultracentrifugation.
Hepatitis B virus core protein (HBc) is an important serology marker of hepatitis B infection and patient follow-up. It is an M, 21,000 protein, which has the intrinsic capacity to self-assemble as a capsid-like particle. The hepatitis B core protein has been expressed in Escherichia coli and Pichia pastoris (three different constructions) in order to select a HBc recombinant antigen suitable for serodiagnosis requirements with a cost effective downstream strategy. The expression and purification of the different forms of recombinant HBc have been described. For the last step, ultracentrifugation and size-exclusion chromatography were compared. The morphology of these capsids was observed using an electron microscope. Our data shows that HBc antigen is produced in large quantities in E. coli but some contaminants remained which were associated with the E. coli HBc protein after ultracentrifugation or size-exclusion chromatography. The ultracentrifugation enables a higher purity of HBc antigen to be obtained than size-exclusion chromatography but the latter enables a higher recovery rate. P. pastoris enables the expression and extraction of a highly purified HBc antigen suitable for diagnostic purposes.
Rolland
D
D
BioMérieux S.A., Marcy-l'Etoile, France.
Gauthier
M
M
Dugua
J M
JM
Fournier
C
C
Delpech
L
L
Watelet
B
B
Letourneur
O
O
Arnaud
M
M
Jolivet
M
M
D002850:Chromatography, Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006512:Hepatitis B Core Antigens / Q000235:genetics / Q000302:isolation & purification*; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D014461:Ultracentrifugation / Q000379:methods*
D006512:Hepatitis B Core Antigens; D011994:Recombinant Proteins