DESIGN, EXPRESSION, AND INITIAL CHARACTERIZATION OF M1, A DE-NOVO PROTEIN ENRICHED IN ESSENTIAL AMINO-ACIDS

Using recently emerging protein folding principles we have designed a protein enriched in the essential amino acids methionine, threonine, lysine and leucine. Our preliminary study of consensus residues (based on charge, hydrophobicity and volume) of natural alpha-helical bundle proteins indicated that the residues M, T, K, and L could be inserted in an alpha-helical bundle structure. We therefore attempted to create a stable de novo protein, highly enriched in these essential amino acids, that would adopt the alpha-helical bundle fold. The design process was an iterative one. The consensus residues (based on the properties profile) for bundle helices were found considering the four helices taken together, helices I to IV individually, or only their N- and C-termini. Using these data, the helices in our de novo protein were designed by inserting tbe residues M, T, K and L as often as possible at positions where their volume, hydrophobicity and charge match the consensus found in natural bundle helices. Short sequences of strong turn formers were used to join the helices and adjust the predicted pI to 7.7, while a number of local and global factors were used to refine our design. Further, the sequence was checked to eliminate various known protease targets in E. coli. The sequence of our de novo protein, MB1, is: MAT-EDMTDMMTTLFKTMQLLTK-SEPTA-MDEATKTATTMKNHLQNLMQK-TKNK-EDMTDMATTYFKTMQLLTK-TEPSA-MDEATKTATTMKNHLQNLMQK-GVA, where dashes separate long helices from short, turn forming linkers. A gene coding for this protein was assembled from synthetic oligonucleotides, then fused to the maltose binding protein gene under the control of a tac promoter. The fusion protein was expressed in E. coli. purified and cleaved to yield maltose binding protein and our de novo protein, MB1. MB1 was found to be helical, to have the expected molecular weight (11 kDa) and the expected content (57%) of the essential amino acids M, T, K and L.