Heterotetramers formed by an S-layer-streptavidin fusion protein and core-streptavidin as a nanoarrayed template for biochip development.
Based on the S-layer protein SbpA of Bacillus sphaericus CCM 2177, an S-layer-streptavidin fusion protein was constructed. After heterologous expression, isolation of the fusion protein, and refolding, functional heterotetramers were obtained that had retained the ability to recrystallize into the square-lattice structure on plain gold chips and on gold chips precoated with secondary cell wall polymer (SCWP), which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Monolayers generated by recrystallization of heterotetramers on plain gold chips or on gold chips precoated with thiolated SCWP were exploited for the binding of biotinylated oligonucleotides (30-mers). Hybridization experiments with complementary fluorescently labeled oligonucleotides carrying one mismatch or no mismatch (both 15-mers) were performed and evaluated with surface-plasmon-field-enhanced fluorescence spectroscopy. For surfaces generated by the recrystallization of heterotetramers on SCWP-coated gold chips, a detection limit of 1.57 pM could be determined, whereas for surfaces obtained by direct recrystallization of heterotetramers on plain gold chips, a detection limit of 8.2 pM was found. Measuring the association and dissociation processes of oligonucleotides carrying no mismatch led to a dissociation constant of K(D)=6.3 x 10(-10) m, whereas for oligonucleotides carrying one mismatch a dissociation constant of K(D)=7.9 x 10(-9) m was determined. This finding was confirmed by measuring the whole Langmuir isotherm, which resulted in a dissociation constant of K(D)=2.6 x 10(-8) m.
Huber
Carina
C
Center for NanoBiotechnology, University of Natural Resources and Applied Life Sciences Vienna, Gregor Mendel Strasse 33, 1180 Vienna, Austria. carina.huber@boku.ac.at
Liu
Jing
J
Egelseer
Eva M
EM
Moll
Dieter
D
Knoll
Wolfgang
W
Sleytr
Uwe B
UB
Sára
Margit
M
D019921:Biotinylation; D003460:Crystallization / Q000379:methods*; D019281:Dimerization; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008422:Materials Testing; D008968:Molecular Conformation; D046912:Multiprotein Complexes / Q000737:chemistry; D049329:Nanostructures / Q000737:chemistry* / Q000648:ultrastructure; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D010316:Particle Size; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry*; D020349:Surface Plasmon Resonance / Q000379:methods*; D013499:Surface Properties
D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; D019809:Streptavidin