Expression, Purification, And Characterization Of Elastin-Like Polypeptides Containing Chondroitin Sulphate Binding Domains

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Date
2013-01-07
Authors
Murphy, Mary
Keyword
Tissue Engineered Blood Vessels (TEBVs) , Hyperplasia , Chondroitin Sulphate , Coacervation , Elastin , Elastin-Like Polypeptides
Abstract
The development of small-diameter artificial blood vessels that mimic the properties of natural blood vessels has proven to be a clinical challenge. While autologous vessels are the standard, they can be difficult to obtain and require invasive surgeries. Synthetic materials have been successful in large diameter applications, but they have been unsuccessful in small-caliber environments due to a number of factors including thrombus formation, intimal hyperplasia, and infection. Intimal hyperplasia, of particular interest in this study, involves the build up of smooth muscle cells (SMCs) in the intimal layer of the artery due to abnormal migration and proliferation. This work focuses on the development of a new polymer that has the potential to function as an intimal/medial component of a small-diameter blood vessel. Using recombinant elastin-like polypeptides (ELPs) developed by the Woodhouse laboratory, as well as chondroitin sulphate-specific binding sequences (CSBD1 and CSBD2) determined by the Panitch laboratory, a new elastin-like polypeptide-chondroitin sulphate binding domain (ELP-CSBD) block copolymer has been developed and characterized. The expression of the ELP1-CSBDs was accomplished using E. coli BL21 cells in a bioreactor or shaker flask systems. The polypeptides were purified using dialysis and ion exchange chromatography and expression and purity were characterized using mass spectrometry and amino acid analysis. Both ELP1-CSBDs were successfully expressed using these methods and ELP1-CSBD1 was produced to high purities. ELP1-CSBD1 was able to undergo coacervation in vitro, suggesting that ELP1-CSBD1 is able to self-assemble in a manner similar to native elastin. In the presence of the glycosaminoglycan chondroitin sulphate (CS), the temperature of coacervation of ELP1-CSBD1 is increased, the rate and extent of coacervation is decreased, and aggregates remain in solution even at higher temperatures. The influence of heparin was also explored as previous studies indicated that the CS binding domains were shown to also bind to heparin. Studies completed in the presence of heparin showed that there were no significant changes to the coacervation characteristics of ELP1-CSBD1. It is anticipated that when combined with CS, ELP1-CSBD1 will gel, forming a basis for an intimal/medial layer of a TEBV that will modulate SMC response and increase graft integrity.
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