Large-surface biosensor technology for enhanced recovery in protein characterization.
A large-surface biosensor technique using surface plasmon resonance (SPR) was tested for protein purification by recovery of a monoclonal antibody against human proinsulin C-peptide. Notably, both reversible attachment/desorption and actual purification of the antibody from a multi-component protein mixture was shown. For initial chip attachment of the peptide ligand, C-peptide was biotinylated and attached to neutravidin on plastic chips with a large gold surface (effective area 26 mm(2)). Antibody binding and desorption was monitored in real-time SPR, and for elution different conditions were employed. Five percent formic acid (in contact with the chip surface for 3 min) in a 60-mul segment between air bubbles was efficient for subsequent analysis. In this manner, protein amounts up to 35 pmoles were recovered in a single capture/elution cycle. Evaluation by SDS-PAGE showed essentially no carryover between fractions in this elution process, and also not with other proteins in the mixture after purification. Compared to existing commercial instruments, this technique gives higher recovery and makes it possible to monitor monitor protein binding/desorption. Recovery of affinity partners at the multi-pmole level is demonstrated for protein purification in SPR approaches.
Melles
Ermias
E
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Bergman
Tomas
T
Ståhlberg
Marie
M
Thirstrup
Carsten
C
Wahren
John
J
Jörnvall
Hans
H
Shafqat
Jawed
J
D000327:Adsorption; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001360:Avidin / Q000493:pharmacokinetics; D015374:Biosensing Techniques; D001709:Biotechnology / Q000379:methods*; D019921:Biotinylation; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry; D008970:Molecular Weight; D011384:Proinsulin / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance / Q000379:methods*; D014357:Trypsin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D008024:Ligands; D011506:Proteins; C093483:neutravidin; D001360:Avidin; D011384:Proinsulin; D014357:Trypsin