Semisynthesis of Hemoglobin

Protein engineering, generation of mutant or modified forms of protein, has become the first step for studying the correlation of structure and function of proteins. Design and generation of novel protein molecules with tailormade properties is the long-range goal of such studies. Such novel protein molecules are now designed and generated by recombinant DNA technology, as long as these protein molecules contain only the naturally occurring 20 amino acids residues (1 ,2 ). However, if unnatural amino acids needs to be introduced, cell-free protein expression system and special manipulation of the tRNA is necessary (3 ,4 ). Incorporation of the unnatural amino acid residues into a protein in a site-specific fashion could also be achieved through total chemical synthesis. Gutte and Merrifield (5 ) achieved the total chemical synthesis of RNase-A using solid-phase synthesis, starting from the carboxyl end of the molecule and building, one residue at a time, to its amino terminus. Hoffman et al. (6 ) introduced an alternate approach that involved the synthesis of a limited number of medium-size, protected segments of the molecule and condensing them to generate the full-length protein. This approach, generally referred to as a segment condensation approach (6 ), has gained significant attention in recent years. The molecular size of the proteins that biochemists desire to design and assemble are larger compared to RNase A. Accordingly, interest in developing newer and simpler approaches of segment condensation has increased.