Molecular Interactions for Nanobiotechnology

Many bacteria and archaea possess crystalline surface layers or S-layers as their outermost cell envelope component. Due to the specific property of S-layer proteins to self-assemble into monomolecular protein lattices, they were exploited for the construction of functional S-layer fusion proteins. For recrystallization of S-layer fusion proteins on solid supprts, the latter were precoated with secondary cell wall polymers (SCWPs), which represent the natural anchoring moleules for many S-layer proteins in the bacterial cell wall. So far, the chimeric genes encoding self-assembling S-layer fusion proteins have been expressed in Escherichia coli. To circumvent problems arising from a gram-negative expression host, a homologous expression system in Bacillus sphaericus CCM 2177 shall be established by inactivating the chromosomal S-layer gene sbpA and replacing it by the sequences encoding rSbpA-fusion proteins. The binding mechanism between S-layer-homologous (SLH)-domains and pyruvylated SCWPs was found to be wide-spread among bacteria. As in the case of the S-layer protein SbpA the SLH-domain was not sufficient for SCWP binding, a further sequence or domain, which together with the SLH-domain forms the functional unit, shall be identified. For that purpose, the sequences encoding C-terminal truncations of the sbpA gene shall be expressed in B. sphaericus CCM 2177 and binding of these rSbpA forms to the SCWP will be investigated by surface plasmon resonance (SPR) measure-ments. Site-specific mutagenesis shall be performed to identify amino acids involved in the binding process.To elucidate the role of the pyruvic acid residues in the SCWP for the attachment of other cell wall-associated exoproteins than S-layer proteins, comparative studies between the B. sphaericus CCM 2177 wild-type strain and a knock-out mutant, in which the gene responsible for SCWP pyruvylation has been deleted, will be carried out. SCWP targeting domains shall be identified by producing N- or C-terminally truncated forms of selected cell-associated exoproteins and by performing SPR studies. The existence of a second conserved binding mechanism between S-layer proteins and other cell wall-associated exoproteins that do not carry an SLH-domain, but possess a certain conserved sequence, and non pyruvylated, (N-acetyl) mannosaminuronic acid-containing SCWPs, shall be investigated. Furthermore, domains responsible for binding of an exoamylase to the S-layer and to the rigid cell wall layer of Geobacillus stearothermophilus ATCC 12980 will be identified. The knowledge on biomolecular interactions shall be exploited for the construction of novel fusion proteins used as patterning elements in nanobiotechnology.

Many bacteria and archaea possess crystalline surface layers or S-layers as their outermost cell envelope component. Due to the specific property of S-layer proteins to self-assemble into monomolecular protein lattices, they were exploited for the construction of functional S-layer fusion proteins. For recrystallization of S-layer fusion proteins on solid supprts, the latter were precoated with secondary cell wall polymers (SCWPs), which represent the natural anchoring moleules for many S-layer proteins in the bacterial cell wall. So far, the chimeric genes encoding self-assembling S-layer fusion proteins have been expressed in Escherichia coli. To circumvent problems arising from a gram-negative expression host, a homologous expression system in Bacillus sphaericus CCM 2177 shall be established by inactivating the chromosomal S-layer gene sbpA and replacing it by the sequences encoding rSbpA-fusion proteins. The binding mechanism between S-layer-homologous (SLH)-domains and pyruvylated SCWPs was found to be wide-spread among bacteria. As in the case of the S-layer protein SbpA the SLH-domain was not sufficient for SCWP binding, a further sequence or domain, which together with the SLH-domain forms the functional unit, shall be identified. For that purpose, the sequences encoding C-terminal truncations of the sbpA gene shall be expressed in B. sphaericus CCM 2177 and binding of these rSbpA forms to the SCWP will be investigated by surface plasmon resonance (SPR) measure-ments. Site-specific mutagenesis shall be performed to identify amino acids involved in the binding process.To elucidate the role of the pyruvic acid residues in the SCWP for the attachment of other cell wall-associated exoproteins than S-layer proteins, comparative studies between the B. sphaericus CCM 2177 wild-type strain and a knock-out mutant, in which the gene responsible for SCWP pyruvylation has been deleted, will be carried out. SCWP targeting domains shall be identified by producing N- or C-terminally truncated forms of selected cell-associated exoproteins and by performing SPR studies. The existence of a second conserved binding mechanism between S-layer proteins and other cell wall-associated exoproteins that do not carry an SLH-domain, but possess a certain conserved sequence, and non pyruvylated, (N-acetyl) mannosaminuronic acid-containing SCWPs, shall be investigated. Furthermore, domains responsible for binding of an exoamylase to the S-layer and to the rigid cell wall layer of Geobacillus stearothermophilus ATCC 12980 will be identified. The knowledge on biomolecular interactions shall be exploited for the construction of novel fusion proteins used as patterning elements in nanobiotechnology.