Indirect immobilization of recombinant proteins to a solid phase using the albumin binding domain of streptococcal protein G and immobilized albumin.
Immobilization of proteins to a solid phase leads to denaturation of the adsorbed molecules which may subsequently affect biological interactions. However, for many applications maintenance of the native structure is desired. Therefore, an indirect immobilization system was developed, based on binding of the albumin binding domain (ABP) of streptococcal protein G to rat serum albumin (RSA) precoated on a solid phase (RSA-microtiter plates). Escherichia coli vectors were adapted for production of recombinant protein fused to ABP and the 6 X His-tag. The expressed ABP tag was found to form homodimers. Plasmon resonance was used to study the interaction between an ABP fusion protein and immobilized RSA. Apparent on- and off-rates were calculated using a model for a bivalent analyte (k(a1) = 3.37 x 10(4) M(-1) s(-1), k(d1) = 1.23 x 10(-4) s(-1)). Thus, the stability of the ABP-RSA interaction can be explained by a slow off-rate. This was confirmed by chase experiments in an ELISA format. The ABP-RSA interaction remained stable after addition of different albumins. This immobilization system was used for the development of an ELISA to detect antibodies against Borna disease virus protein p40. The use of RSA-microtiter plates for indirect immobilization of ABP fusion protein was shown to be superior to direct adsorption on plastic. To obtain maximal antibody binding ten times less antigen was needed for indirect immobilization compared to direct adsorption. The binding capacity of the RSA-microtiter plates was determined to be about 0.8 pmol of monomeric ABP protein.
Baumann
S
S
Institute of Virology, University of Zürich, Switzerland.
Grob
P
P
Stuart
F
F
Pertlik
D
D
Ackermann
M
M
Suter
M
M
D000327:Adsorption; D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis; D000956:Antigens, Viral / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D001891:Borna disease virus / Q000276:immunology; D004355:Drug Stability; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism
D000914:Antibodies, Viral; D000956:Antigens, Viral; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D011994:Recombinant Proteins; D012709:Serum Albumin