Faculty: Dr. C. Filipe

 

Major Research Interests

Our major research interests are in the area of applied molecular biology. Our current focus in research is on:

·        Development of simple of techniques for purification of recombinant proteins.

·        In vivo creation of artificial compartments in E. Coli

·        Development of methods for expression and purification of membrane proteins. In this area, we are experimenting with generation of self-assembled membrane like structures in the cytoplasm of E. Coli

·        We recently started working on methods to stabilize messenger RNA, as well as methods to increase the readability of transcripts, with the objective of enhancing control on gene expression.

Representative Example of a Research Project

Recombinant proteins are produced for a variety of applications, such as for enzyme production, therapeutics, proteomics, structural studies, and many other areas. The need for these proteins therefore ranges from the very small scale (such as in proteomics and in assays) to very large scale industrial processes. We developed a very simple method to purify recombinant proteins by fusing a target protein with an intein and an elastin-like polypeptide that only requires NaCl, dithiothreitol, and a syringe filter to isolate the target protein from Escherichia coli lysate (J. Am. Chem. Soc.; 2005; 127(32); 11228-11229). The tripartite   

 

 

Figure: Tripartite fusion system for simple recombinant purification.

 

 

This tripartite fusion system enables rapid isolation of the target protein without the need for affinity chromatography for purification or proteases for cleavage of the target protein from the fusion. The elastin-like polypeptide tag imparts reversible phase transition behavior to the tripartite fusion so that the fusion protein can be selectively aggregated in cell lysate by the addition of NaCl. The aggregates are isolated by microfiltration and resolubilized by reversal of the phase transition in low ionic strength buffer. After resolubilizing the fusion protein, the intein is activated to cleave the target protein from the elastin-intein tag, and the target protein is then isolated from the elastin-intein fusion by an additional phase transition cycle. The following diagram shows this sequence of steps.

 

This work has been highlighted as a technology concentrate in Chemical and Engineering News and in the magazine The Scientist.

 

Representative Publications

Ge, X., Trabbic-Carlson, Chilkoti A., Filipe C.D.M. (2006). Purification of an elastin-like fusion protein by microfiltration. Biotechnology and Bioengineering, 95, 424-432.

Ge, X., Trabbic-Carlson, Chilkoti A., Filipe C.D.M. (2006). Simultaneous phase transition of ELP tagged molecules and free ELP: An efficient and reversible capture system. Biomacromolecules, 7, 2475-2478.  

Su., S., Nutiu, R., Filipe, C.D.M., Li, Y., Pelton, R.(2006). Adsorption and Covalent Coupling of an ATP-binding DNA Aptamers onto Cellulose. Langmuir in press.  

Ge, X., Yang, D.S., Trabbic-Carlson, K., Kim, B., Chilkoti A., Filipe C.D.M. (2005) Self-cleavable stimulus responsive tags for protein purification without chromatography. Journal of the American Chemical Society, 17, 11228-11229  

Filipe, C. D. M., Ghosh, R. (2005). Effects of protein-protein interaction in ultrafiltration based fractionation processes. Biotechnology and Bioengineering, 91678-687.  

Kanani D., Ghosh, R., Filipe, C. D. M., (2004). A novel approach for high-resolution protein–protein separation by ultrafiltration using a dual-facilitating agent . Journal of Membrane Science, 243, 223-228

Filipe, C.D.M., Daigger, G.T. and Grady, C.P.L. Jr. (2001).  A metabolic model for acetate uptake under anaerobic conditions by glycogen accumulating organisms: stoichiometry, kinetics, and the effects of pH.  Biotechnology and Bioengineering, 76, 17-31.

Filipe, C.D.M., Daigger, G. T. and Grady, C.P.L. Jr (2001). Stoichiometry and kinetics of acetate uptake under anaerobic conditions by an enriched culture of phosphorus accumulating organisms at different pHs.  Biotechnology and Bioengineering, 76, 32-43.

Filipe, C.D.M., Daigger, G.T. and Grady, C.P. L. Jr (2001)  Effects of pH on the rates of aerobic metabolism of phosphate accumulating and glyogen accumulating organisms.   Water Environment Research, 73, 213-222.

Filipe, C.D.M., Daigger, G.T. and Grady, C.P. L. Jr (2001). pH as a Key Factor in the competition between Glycogen Accumulating and Phosphorus Accumulating Organisms. Water Environment Research, 73, 223-232.