Assumptions for Protein Engineering,Domain Structures,Synthetic Fragment

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	Assumptions for Protein Engineering - While attempting protein engineering, one should recognize the following properties of enzymes: (i) many amino acid substitutions, deletions or additions lead to no change in enzyme activity, so that they are silent mutations; (ii) proteins have a limited number of basic structures and only minor changes are superimposed on them leading to variation; (iii) similar patterns of chain folding and domain structure can arise from different amino acid sequences, which show little or no homology (although same amino acid sequence never gives different folding and domain structures).
The above properties suggest that while many major changes sometimes may lead to no alteration in function, some of the minor changes at specific positions may lead to the desired favourable change. For example, a single amino acid replacement (glycine to aspartic acid) in E. coli asparate transcarbamylase leads to (i) loss of activity and to (ii) an alteration in the binding of catalytic and regulatory subunits. Another example involved the engineering of a single chain biosynthetic antibody binding site (BARS), which is though only one sixth of the size of the complete antibody, but retains its antigen binding specificity.
This synthetic fragment has heavy and light chain variable regions (V H and V J connected by a 15 - amino acid linker. A synthetic gene has also been prepared for the fragment, which expressed in E. coli. This fragment binds to digoxin, a cradiac glycoside. Single amino acid replacements in BABS fragment have sometimes led to major changes in its binding affinity. In view of the above, it is necessary to examine not only the crystal structure but also the active sites therein, so that the gene may be modified or artificially synthesized for protein engineering to meet the desired needs.