The overall objective of this competing R01 renewal is the continued development of the Elastin Fusion Protein technology as a genetically encodable, biomimetic analog of -""""""""smart"""""""" protein-polymer conjugates. The overall objective is motivated by the rationale that simple and convenient methods to modulate the physico-chemical properties of proteins -most notably their solubility in solution and binding to a surface- in response to an external trigger have immediate application in many areas of Bioengineering and Biotechnology such as protein purification, affinity capture, protein chips, lab-on-a-chip bioanalysis and in the development of environmentally responsive biomaterials and nanocarriers for delivery of protein therapeutics. The objective of the proposed research will be accomplished by the gene-level fusion of different recombinant proteins to a stimulus-responsive elastin-like polypeptide (ELP) tag to create ELP fusion proteins. The overall hypothesis of this research -validated for a set of proteins in the previous funding period- is that environmentally sensitive and reversible solubility and binding to a surface can be imparted to any soluble protein of interest by N-or C-terminal fusion to an ELP sequence. The proposed research has two research foci: the first, """"""""fundamental focus"""""""", seeks to discover the biophysical basis of the effect that fusion of proteins has on the inverse transition behavior of an ELP. The molecular level understanding of the inverse transition behavior of ELP fusion proteins will directly feed into the second, """"""""technological focus"""""""" that will develop an ensemble of applications that exploit the inverse transition behavior of ELP fusion proteins. The second focus consists of three related technological applications: (1) optimization of the expression and purification of ELP fusion proteins in a bacterial expression system; (2) affinity capture of biomolecules by an ELP fusion protein followed by their separation by inverse transition cycling; and (3) """"""""direct, on chip"""""""", reversible capture of ELP fusion proteins from solution on surfaces functionalized with ELPs. ? ?
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