For several other case studies about rational protein designing students may read following article.

Protein Engineering BY DIRECTED EVOLUTION

Let us assume a condition where a researcher has to design a protein which binds best with a given receptor or biomolecule. Let us see how directed evolution can help in this designing.

Phage display: George Smith pioneered phage-display technology in 1985. It was reported that a phage displaying the foreign peptides were able to infect bacteria in the same manner as wild type and fusion protein was functional. Phage display is a powerful method for engineering proteins with desired binding specificities. In this method M13, Escherichia coli -specific filamentous bacteriophage (a virus infecting bacteria) is used. G ene fragments encoding polypeptides or a peptide library are fused to M13 coat protein genes. This fusion protein becomes part of the capsid and the heterologous protein is displayed on the surfaces of the phage. M13 filamentous phage, contain a circular single stranded DNA genome. The genome encodes 10 proteins, 5 of which are virion structural proteins. The genome is enclosed in a protein coat encoded predominantly by 2700 copies of gene protein VIII (gpVIII) which constitute the major coat protein.

At the 'tail' end of the phage particle 4-5 copies of the gene VII protein (gpVII) and 4-5 copies of the gene IX protein (gpIX) are found and they are involved in initiating phage assembly and maintaining the stability of the viral particle. At the other end of the phage particle are 3-5 copies of the gene III protein (gpIII) which is required for infection of the host cell and 4-5 copies of the gene VI protein (gpVI) involved in the termination of the viral assembly process. Although gpIII is used for making fusion construct but recently gpVII and gpIX have also been shown to tolerate fusions at their amino termini (Fig. 3).