categories: method development, biomaterials, high-throughput screening, silk-elastin-like proteins
Qin Wang, Xiaoxia Xia, Wenwen Huang, Yinan Lin, Qiaobing Xu,* and David L. Kaplan*
Department of Biomedical Engineering, Tufts University, Massachusetts, USA and State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Advanced Functional Materials, 2014, in press, doi: 10.1002/adfm.201304106
Highlight by: Dr. Alessandro Poma, The Open University
This study describes for the first time the construction and screening of a Silk-Elastin-Like Protein (SELP) library in which different amino acids were tested at the “X” position of the elastin domain (thus obtaining a broader range of functional material properties) and systematically compared in terms of dynamic properties (thermal, electrochemical, mechanical and adhesive) using high-throughput methods. The aim of the authors was to improve the fundamental understanding of the stimuli-responsive properties of silk and elastin, together with the fulfillment of the need for more elastic polymeric systems in many biomaterial applications.
Central to the work is the generation of 12 monomeric genes and subsequent engineering of plasmids and transformation to encode SELP polymers in E. coli host cells. The colonies containing plasmids carrying SELP genes in various lengths or empty plasmids were randomly cultured in 96 well plates and the expressed protein polymer library was purified in situ through inverse temperature transition cycling and screened by optical response, thus selecting 64 SELPs from the initial screening of over 2000 colonies. The selected proteins (ranging from 20 to 130 kDa in molecular weight) were further characterized and selected for specific functions by screening for optical, mechanical or adhesive properties in dependence of which amino acid was present at the “X” position of the elastin domain or in dependence of the “elastin-to-silk” ratio in the polymer. The results obtained suggest that the hydrophobicity or hydrophilicity of the amino acid in position X greatly influences the thermal response of the polymer, as well as its adhesive properties, especially for high molecular weights, while its elastic module is more affected by the “elastin-to-silk” ratio.
This work directly addresses the lack of diversity of current protein polymers as biomaterials due to the low efficiency of synthesis and limited nature of screening procedures currently used. The combinatorial methods described here can be extended to construct libraries for other protein motifs (e.g., collagens, keratins, binding domains).
- Peptide-based biopolymers reviews: (a) “Peptide-based Biopolymers in Biomedicine and Biotechnology” Chow, D.; Nunalee, M.L.; Lim, D.W.; Simnick, A.J.; Chilkoti, A., Mater. Sci. Eng. R. Rep. 2008, 62, 125-155. (b) “Peptide-based fibrous biomaterials: some things old, new and borrowed” Woolfson, D.N.; Ryadnov, M.G., Curr. Opin. Chem. Biol. 2006, 10, 559-567.
- Silk-elastin-like proteins: (a) “Genetic engineering of stimuli-sensitive silkelastin-like protein block copolymers.” Nagarsekar, A.; Crissman, J.; Crissman, M.; Ferrari, F.; Cappello, J.;, Ghandehari, H. Biomacromolecules 2003, 4, 602-607. (b) “A Complete Recombinant Silk-Elastinlike Protein-Based Tissue Scaffold” Qiu, W.; Huang, Y.; Teng, W.; Cohn, C.M.; Cappello, J.; Wu, X., Biomacromolecules 2010, 11, 3219-3227.