Enhanced Recovery and Folding of Recombinant Proteins Using Fusion Protein Strategies
In protein chemistry, significant efforts have been made trying to solve how information from the amino acid sequence is transferred into a three dimensional structure. The understanding of the protein folding problem has increased in recent years. From the beginning the topic was primarily of academic interest but has now become an issue of industrial importance. Production of recombinant proteins in heterologous hosts often lead to unfolded and inactive proteins, in particular when the production levels are high. In order to generate useful protein products it is necessary to refold the protein in vitro. The folding problem was elegantly outlined by Levinthal and is referred to as "the Levinthals paradox" (Levinthal, 1969): Assume a protein with 100 amino acids where every residue can have two different conformations. Despite this very simplified model, the protein can adopt 1~ different conformations. Even if the protein is able to sample new configurations at the rate of 1015 per second it will need 3 x 109 years to fold, similar to the age of earth. The true allowed number of conformations for an amino acid is of course much higher than two, and the time for a protein to fold completely random to search for the native conformation must exceed life of the earth. In contrast to these statistical figures, folding of an a-helix is estimated to take place in a time scale of 1o-6 seconds (Creighton, 1993). Consequently, there must be a non-random pathway for a protein to fold into an active structure.