A New Approach to Artificial and Modified Proteins: Theory-based Design, Synthesis in a Cell-free System and Fast Testing of Structural Properties by Radiolabels

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A novel approach to the creation of artificial and modified proteins has been elaborated. The approach includes a sequence design based on the molecular theory of protein secondary structure and folding patterns, gene expression in a cell-free system and testing of structural properties of the synthesized polypeptides at a nanogram level using radiolabelled chains. The approach has been applied to a new synthetic protein albebetin which has been designed to form a 3-D fold which does not contradict any structural rule but has been never observed up to now in natural proteins. Using size-exclusion chromatography, urea-gradient electrophoresis and limited proteolysis of a radiolabelled chain, it has been shown that the artificial protein is nearly as compact as natural proteins, cooperatively unfolds at high urea concentrations and has some structural features of a definite structure consistent with the designed one. As albebetin has been designed as consisting of two structural repeats, a ‘halfalbebetin’ (one of these repeats) has also been synthesized and studied. It was shown that ‘half-albebetin’ is also compact

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Protein Engineering, Design and Selection, v. 7, issue 8, p. 1041-1052