The coiled coils in the design of protein-based constructs:

hybrid hydrogels and epitope displaysAijun 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,

USAbDepartment of Bioengineering , 20S 2030E Rm. 205,University of Utah , Salt Lake City

,UT 84112,USA

Published in Journal of Controlled Release 72(2001)

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

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

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

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

Self replicating peptide

Coiled Coils

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

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

Design of hybrid hydrogels using coiled coil motif

T increase

Design of an epitope display system usingcoiled 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.

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

BiomoleculesApplication

Biosensing

Affinity Separation

Generating surfaces with specific biocompatibility

Design of an epitope display system usingcoiled coil motif

Immobilization Technique

Physical Adsorption

Covalent Binding

Langmuir-Blodgett tech (LB) : popular for ordered monolayers of amphipiles

SAM

Alkanethiolate SAMs

Alkyl Siloxane SAMs

Design of an epitope display system usingcoiled 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

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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