No abstract

A simple technique is presented for non-chromatographic purification of recombinant proteins expressed in Escherichia coli. This method is based on a reversibly precipitating, self-cleaving purification tag. The tag is made up of two components: an elastin-like polypeptide (ELP), which reversibly self-associates in high-salt buffers at temperatures above 30 degrees C; and an intein, which causes the ELP tag to self-cleave in response to a mild pH shift. Thus, a tripartite ELP-intein-target protein precursor can be purified by cycles of salt addition, heating and centrifugation. Once purified, intein-mediated self-cleavage, followed by precipitation of the cleaved ELP tag, allows easy and effective isolation of the pure, native target protein without the need for chromatographic separations. Recoveries of 50-100 mg of cleaved, native target protein per liter of shake-flask culture have been achieved for over a dozen proteins, typically in 8-24 h depending on specific process parameters.

The development of waxy crude oil and some gas condensate fields can lead to serious operational problems because of solidification of the paraffin components of the fluid in flowlines. Many numerical models in the literature predict the thickness of the wax deposit. However, most of these models assume that the wax-oil (gel) deposit has a constant wax content. In this work, we analyze wax deposition in laminar flow regime to predict the thickness and the composition of the gel layer as a function of position and time. The wax-oil gel region is considered as a porous medium. The velocity field and the pressure drop are calculated from the Navier−Stokes equation in the liquid region and from a combined Darcy-type equation and the Navier−Stokes equation in the gel region. The wax amount is estimated as a result of a decrease in fluid temperature below the wax appearance temperature (WAT), counterdiffusion processes from thermal and molecular diffusions, and radial convection which occurs because of nonuniform gel layer thickness. We compare predicted results from our model with several experimental data from the literature. The results which are in agreement with data cannot be predicted by formulations in which chain rule is used to replace concentration gradient with temperature gradient in the molecular diffusion expression.