1. The coiled coils in the design of protein-based constructs: hybrid hydrogels and epitope displays Aijun Tang a, Chun Wang b, Russell J. Stewart b, Jindrich Kopecek a, b* aDepartment of Pharmaceutics and Pharmaceutical Chemistry, 30S 2000E Rm. 301,University of Utah, Salt Lake City,UT 84112, USA bDepartment of Bioengineering, 20S 2030E Rm. 205,University of Utah, Salt Lake City,UT 84112,USA Published in Journal of Controlled Release 72(2001)

2. Introduction Protein-based biomaterials and biomaterials with Proteins have been attracting more attention recently Protein polymers contain mixtures of molecules with different chain length, lack of control of genetic engineering and chain architecture Genetic Engineering made possible to produce peptide/protein with well defined amino acid sequence and precisely controlled macromolecular architectures including size, composition, sequence

3. Introduction Genetic Engineering Plasmid Technology Engineered protein with non natural function Produce new protein with predetermined properties Conjugation of Polymer with genetically engineered site of protein for wide range purposes

4. Coiled Coils A coiled coil is a bundle of two or more right-handed amphiphilic a-helices wrapping around each other into a slightly left-handed super-helix (Fig. 1A). Consists of heptad repeats[(abcdefg)x] a,d positions occupied by hydrophobic residues The other positions usually occupied by polar ones Hydrophobic interaction stabilizing the coiled coil conformation Heterodimeric coiled coil used in biosensor and affinity purification

5. Coiled Coils Parallel and antiparallel determined by electrostatic interaction between residues e and g Affinity matrix for protein purification & Biosensor Application Multivalent Antibody Conformationally defined synthetic lybray

6. Self replicating peptide Coiled Coils

7. Design of hybrid hydrogels using coiled coil motif Hydrogels are 3D polymer network that do not dissolve in water but are able to swell and retain significant amount of water, used in surgical sutures, soft tissue prostheses, drug delivery system, soft contact lenses Old Tech Hydrogel Lack exast control on 3D structure Heterogeneity in structure and physicochemical properties Hybrid hydrogel : hydrogel system contained two or more components of dinstinct classes of molecules

8. Design of hybrid hydrogels using coiled coil motif Advantages of Hybrid hydrogel We can combined good properties from different components to achieve a better one, example : assembling Hybrid hydrogel from synthetic polymers and Genetically engineered protein It maybe possible to create HH that are responsive to a variety of stimuly such as ph, ionic strength, solvent, light, mechanical force, and specific ligand

9. Design of hybrid hydrogels using coiled coil motif T increase

10. Design of an epitope display system using coiled coil motif An epitope is the part of a macromolecule that is recognized by the immune system, specifically by antibodies, B cells, or cytotoxic T cells.macromolecule immune systemantibodiesB cellscytotoxic T cells Small synthetic epitopes may represent a new category of targetting moieties for polymer based targettable drug delivery systems, but the problem is we need to find structure biorecognition relationship to achieve best targetting ability Biomolecules Application Biosensing Affinity Separation Generating surfaces with specific biocompatibility

11. Design of an epitope display system using coiled coil motif Immobilization Technique Physical Adsorption Covalent Binding Langmuir-Blodgett tech (LB) : popular for ordered monolayers of amphipiles SAM Alkanethiolate SAMs Alkyl Siloxane SAMs

12. Design of an epitope display system using coiled coil motif Biorecognition sites in proteins are often presented on the surfaces with the whole molecules serving as conformational scaffolds. To mimic natural proteins, protein/peptide construsts, such as coiled coil peptides, can be designed de novo and used a sequence simplified scaffolds into which recognition elements from naturally occuring proteins and peptides can be incorporated

18. Conclusions Applying genetic engineering techniques to biomaterials and drug delivery research offers numerous opportunities of creating protein based constructs with well-defined structure and unique properties Genetically engineered CCP motifs were used to form crosslinks of HH responsive to external stimuli Self Assembly CC stem loop constructs were prepared for studying the biorecognition between ligands and cell surface reseptors This approaches will lead to the development of intelligent drug delivery/release systems and will provide new insight for the rational design of more effective targetable drug carriers

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