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Creative Biolabs can assist customers’ immunity protein 9 research through the high quality library construction service. With years of research and development experience in library construction realm, our scientists are confident in providing high quality, diversity and affinity libraries for customers’ individual requirements.
The immunity protein 9 (Im9) is an acidic immunity protein that belongs to a group of immunity proteins which shares up to 50% sequence identity and can tightly bind to their respective DNase colicins (bacteriocins). For instance, Im9 has been confirmed to form a KD 9.3×10-17 M complex with the DNase of colicin E9 (ColE9), which is known as one of the highest affinities for a protein-protein interaction (Wallis et al. 1995). Im9 is produced by enterobacteria for nonspecific endonucleolytic cleavage of single- and double-stranded DNA of competing bacteria. In the natural role, Im9 binds to the basic cytotoxic domain of colicin, the complexation of Im9 with ColE9 neutralizes the cytotoxic activity of the latter within the producing cell.
As an extensively studied protein, Im9 is also used as scaffold to develop unconventional binding ability. Structurally, Im9 is a 9.5 kDa novel all-α-coil scaffold, which consists of a distorted, antiparallel four α-helical bundle structure with prematurely truncated helix III by a proline residue (Pro 56). Protein engineering experiments have shown that residues in helix III (residues 51-55) are very important for DNase binding, particularly Tyr 54 and Tyr 55. Meanwhile, according to the high affinity achieved by Im9-ColE9 complex, Im9 is considered as a source to improve highly efficient toxin inhibitor protein. By constructing Im9 scaffold libraries carry appropriate mutations, novel DNase colicin inhibitor can be selected. In fact, Im9 variant was firstly selected by in vitro compartmentalization (IVC) for inhibition of the colicin E7 (ColE7). Moreover, Im9 scaffold libraries are also potential to be involved in other objectives, such as the design of artificial enzymes.
Scientists of Creative Biolabs have developed the novel Hi-Affi™ phage display platform for our library construction services. This platform is an improved exogenous gene expression method upgraded from the traditional phage display technology, a method for displaying interest target on the surface of bacteriophage coat proteins. Compared with traditional phage display method, Hi-Affi™ phage display platform has adopted trimer codon technology and NNK method to improve the library diversity of specific scaffolds. Through this platform, we can ensure the generated scaffold libraries with 100% precise mutant and over 1010 diversity.
With the unparalleled expertise in in the field of scaffold library construction, Creative Biolabs is committed to offer faster and more efficient services for our global customers. We have generated more than 55 different main kinds of scaffold libraries under various conditions. Meanwhile, our scientists are pleased to guarantee the reliability of each service through our strict quality control standards.
Fig. 1 Structure of the E9 DNase (red) bound to Im9 (yellow) at 1.7 Å superimposed on the original structure reported by Kleanthous et al. (1999) at 2.05 Å (grey). (Kühlmann et al. 2000)
Fig. 2 Crystal structure of colicin E9 DNase domain with its cognate immunity protein Im9 (1.7 Å). (PDB ID: 1EMV)
Kleanthous, C., Kühlmann, U. C., Pommer, A. J., Ferguson, N., Radford, S. E., Moore, G. R., James, R. and Hemmings, A. M. (1999) 'Structural and mechanistic basis of immunity toward endonuclease colicins', Nature Structural & Molecular Biology, 6(3), 243-252.
Kühlmann, U. C., Pommer, A. J., Moore, G. R., James, R. and Kleanthous, C. (2000) 'Specificity in protein-protein interactions: the structural basis for dual recognition in endonuclease colicin-immunity protein complexes', Journal of molecular biology, 301(5), 1163-1178.
Wallis, R., Moore, G. R., James, R. and Kleanthous, C. (1995) 'Protein-protein interactions in colicin E9 DNase-immunity protein complexes. 1. Diffusion-controlled association and femtomolar binding for the cognate complex', Biochemistry, 34(42), 13743-13750.
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