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Recover desirable proteins from biologically derived solutions and suspensions

Objective:

Optimally separate and recover a desirable protein from E. coli fermentation broth in one-step and from transgenic goat milk in a pre-filtration step (microfiltration) and a concentration step (ultrafiltration, expanded bed adsorption).

Approach 1:

Use controllable (pH, T) intervening protein sequences (inteins) as cleavable linkers in tripartite fusions (binding domain-linker-product) to recover a desirable protein from a fermentation broth using affinity fusion adsorption in laboratory and fluidics formats (with collaborators Marlene Belfort and Vicky Derbyshire both from the Wadsworth Center, NY State Dept. Health, Albany, NY). See Figure 6 and Figure 7.
Study intein cleavage using molecular simulation calculations (molecular modeling; ab inito calculations) of intein (linker) cleavage and splicing (with collaborators Shekhar Garde and Saroj Nayak from Chemical Engineering and Physics Departments, RPI, respectively).
Use fluorescent quenching and a quartz crystal microbalance to follow kinetics of the cleaving and splicing reaction.

Approach 2:

Recover with high yield a desired protein from transgenic goat milk using microfiltration with control of transmembrane pressure, solution pH, temperature and flow conditions. See Figure 8 and Figure 9.
Concentrate and purify a desired protein from milk with high purity via ultrafiltration or expanded bed adsorption using solution conditions and other operating conditions to advantage.

Relevant Publications:

Wood, D., Wu, W., Belfort, G., Derbyshire V. and Belfort M. (1999) A genetic system to modulate intein function for use in biotechnology, Nature Biotechnology 17, 889-892.
Wood, D., Derbyshire V. Wu, W., Chartrain, M, Belfort, M., and Belfort G. (2000) Optimized Single-Step Affinity Purification with a Self-Cleaving Intein Applied to a Human Fibroblast Growth Factor, Biotechnology Progress 16, 1055-1063.
Wu, W, Wood, D. W., Belfort, G., Derbyshire V., and Belfort, M. (2002) Intein-mediated purification of cytotoxic endonumclease I-Tev1 by insersional activation and pH-controllable splicing, Nuclei Acids Res., 30, 4864-4871.

Sample results:

See Figures 6 - 9