Construction of a functional S-layer fusion protein comprising an immunoglobulin G-binding domain for development of specific adsorbents for extracorporeal blood purification.
The chimeric gene encoding a C-terminally-truncated form of the S-layer protein SbpA from Bacillus sphaericus CCM 2177 and two copies of the Fc-binding Z-domain was constructed, cloned, and heterologously expressed in Escherichia coli HMS174(DE3). The Z-domain is a synthetic analogue of the B-domain of protein A, capable of binding the Fc part of immunoglobulin G (IgG). The S-layer fusion protein rSbpA(31-1068)/ZZ retained the specific properties of the S-layer protein moiety to self-assemble in suspension and to recrystallize on supports precoated with secondary cell wall polymer (SCWP), which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Due to the construction principle of the S-layer fusion protein, the ZZ-domains remained exposed on the outermost surface of the protein lattice. The binding capacity of the native or cross-linked monolayer for human IgG was determined by surface plasmon resonance measurements. For batch adsorption experiments, 3-microm-diameter, biocompatible cellulose-based, SCWP-coated microbeads were used for recrystallization of the S-layer fusion protein. In the case of the native monolayer, the binding capacity for human IgG was 5.1 ng/mm(2), whereas after cross-linking with dimethyl pimelimidate, 4.4 ng of IgG/mm(2) was bound. This corresponded to 78 and 65% of the theoretical saturation capacity of a planar surface for IgGs aligned in the upright position, respectively. Compared to commercial particles used as immunoadsorbents to remove autoantibodies from sera of patients suffering from an autoimmune disease, the IgG binding capacity of the S-layer fusion protein-coated microbeads was at least 20 times higher. For that reason, this novel type of microbeads should find application in the microsphere-based detoxification system.
Völlenkle
Christine
C
Center for Ultrastructure Research and Ludwig Boltzmann Institute for Molecular Nanotechnology, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
Weigert
Stefan
S
Ilk
Nicola
N
Egelseer
Eva
E
Weber
Viktoria
V
Loth
Fritz
F
Falkenhagen
Dieter
D
Sleytr
Uwe B
UB
Sára
Margit
M
D001327:Autoimmune Diseases / Q000276:immunology / Q000628:therapy; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003460:Crystallization; D004274:DNA, Recombinant / Q000235:genetics; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification* / Q000378:metabolism*; D007164:Immunosorbents; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D008863:Microspheres; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D007074:Immunoglobulin G; D007164:Immunosorbents; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C055764:S-layer proteins