Monitoring the production of inclusion bodies during fermentation and enzyme-linked immunosorbent assay analysis of intact inclusion bodies using cryogel minicolumn plates.
A novel minicolumn chromatographic method to monitor the production of inclusion bodies during fermentation and an enzyme-linked immunosorbent assay (ELISA) system allowing direct analysis of the particles with surface-displayed antigens are described. A 33-kDa protein containing 306 amino acids with three sulfur bridges produced as inclusion bodies was labeled with polyclonal antibodies against 15 amino acid (anti-A15) and 17 amino acid (anti-B17) residues at the N- and C-terminal ends of the protein, respectively. Labeled particles were bound to macroporous monolithic protein A-cryogel adsorbents inserted into the open-ended wells of a 96-well plate (referred to as protein A-cryogel minicolumn plate). The concept behind this application is that the binding degree of inclusion bodies from lysed fermentation broth to the cryogel minicolumns increases with an increase in their concentration during fermentation. The technique allowed us to monitor the increase in the production levels of the inclusion bodies as the fermentation process progressed. The system also has a built-in quality parameter to ensure that the target protein has been fully expressed. Alternatively, inclusion bodies immobilized on phenyl-cryogel minicolumn plate were used in indirect ELISA based on anti-A15 and anti-B17 antibodies against terminal amino acid residues displayed on the surface of inclusion bodies. Drainage-protected properties of the cryogel minicolumns allow performance of successive reactions with tested immunoglobulin G (IgG) samples and enzyme-conjugated secondary IgG and of enzymatic reaction within the adsorbent.
Ahlqvist
Josefin
J
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, SE-221 00 Lund, Sweden.
Dainiak
Maria B
MB
Kumar
Ashok
A
Hörnsten
E Gunnar
EG
Galaev
Igor Yu
IY
Mattiasson
Bo
B
D000327:Adsorption; D000907:Antibodies, Bacterial; D019149:Bioreactors; D001798:Blood Proteins; D002845:Chromatography / Q000295:instrumentation / Q000379:methods*; D059985:Cryogels; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000254:growth & development / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000276:immunology; D005285:Fermentation; D005353:Fibronectins; D005434:Flow Cytometry; D020100:Hydrogels; D002479:Inclusion Bodies / Q000378:metabolism* / Q000648:ultrastructure*; D008855:Microscopy, Electron, Scanning; D013205:Staphylococcal Protein A
D000907:Antibodies, Bacterial; D001798:Blood Proteins; D059985:Cryogels; D029968:Escherichia coli Proteins; D005353:Fibronectins; D020100:Hydrogels; D013205:Staphylococcal Protein A