Reversible immobilization of proteins with streptavidin affinity tags on a surface plasmon resonance biosensor chip.
Dissociation of biotin from streptavidin is very difficult due to their high binding affinity. The re-use of streptavidin-modified surfaces is therefore almost impossible, making devices containing them (e.g. surface plasmon resonance (SPR) sensor chips) expensive. This paper describes a new protocol for reversible and site-directed immobilization of proteins with streptavidin affinity tags on the streptavidin-coated SPR biosensor chip (SA chip). Two streptavidin affinity tags, nano-tag and streptavidin-binding peptide (SBP tag), were applied. They both can specifically interact with streptavidin but have weaker binding force compared to the biotin-streptavidin system, thus allowing association and dissociation under controlled conditions. The SA chip surface could be regenerated repeatedly without loss of activity by injection of 50 mM NaOH solution. The fusion construct of a SBP tag and a single-chain antibody to mature bovine prion protein (scFv-Z186-SBP) interacts with the SA chip, resulting in a single-chain-antibody-modified surface. The chip showed kinetic response to the prion antigen with equilibrium dissociation constant K (D) approximately equal to 4.01 x 10(-7). All results indicated that the capture activity of the SA chip has no irreversible loss after repeated immobilization and regeneration cycles. The method should be of great benefit to various biosensors, biochips and immunoassay applications based on the streptavidin capture surface.
Li
Yong-Jin
YJ
Joint research group on analytical biotechnology, State Key Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Bi
Li-Jun
LJ
Zhang
Xian-En
XE
Zhou
Ya-Feng
YF
Zhang
Ji-Bin
JB
Chen
Yuan-Yuan
YY
Li
Wei
W
Zhang
Zhi-Ping
ZP
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002417:Cattle; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011328:Prions / Q000737:chemistry*; D040081:Protein Array Analysis / Q000379:methods*; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry*; D020349:Surface Plasmon Resonance / Q000379:methods*; D013499:Surface Properties; D013997:Time Factors
D007135:Immunoglobulin Variable Region; D010455:Peptides; D011328:Prions; D019809:Streptavidin