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The implementation of generic and efficient technologies for the production of recombinant eukaryotic proteins remains an outstanding challenge in structural genomics programs. We have recently developed a new method for rapid identification of soluble protein expression in E. coli, the colony filtration blot (CoFi blot). In this study, the CoFi blot was used to screen libraries where the N-terminal translation start point was randomized. To investigate the efficiency of this strategy, we have attributed a large number of proteins to this process. In a set of 32 mammalian proteins, we were able to double the success rate (from 34 to 68%) of producing soluble and readily purifiable proteins in E. coli. Most of the selected constructs had their N-termini close to predicted domain borders and the method therefore provides a mean for experimental "domain foot printing." Surprisingly, for most of the targets, we also observed expressing constructs that were close to full-length. In summary this strategy constitutes a generic and efficient method for producing mammalian proteins for structural and functional studies., ((c) 2006 Wiley-Liss, Inc.)

The implementation of efficient technologies for the production of recombinant mammalian proteins remains an outstanding challenge in many structural and functional genomics programs. We have developed a new method for rapid identification of soluble protein expression in E. coli, based on a separation of soluble protein from inclusion bodies by a filtration step at the colony level. The colony filtration (CoFi) blot is very well suited to screen libraries, and in the present work we used it to screen a deletion mutagenesis library. [ABSTRACT FROM AUTHOR]

Thesucker ring teeth (SRT) of Humboldt squid exhibit mechanicalproperties that rival those of robust engineered synthetic polymers.Remarkably, these properties are achieved without a mineral phaseor covalent cross-links. Instead, SRT are exclusively made of silk-likeproteins called “suckerins”, which assemble into nanoconfinedβ-sheet reinforced supramolecular networks. In this study, threestreamlined strategies for full-length recombinant suckerin proteinproduction and purification were developed. Recombinant suckerin exhibitedhigh solubility and colloidal stability in aqueous-based solvents.In addition, the colloidal suspensions exhibited a concentration-dependentconformational switch, from random coil to β-sheet enrichedstructures. Our results demonstrate that recombinant suckerin canbe produced in a facile manner in E. coliand processed from mild aqueous solutions into materials enrichedin β-sheets. We suggest that recombinant suckerin-based materialsoffer potential for a range of biomedical and engineering applications. [ABSTRACT FROM AUTHOR]