In protein engineering

Figure 18.11 Electron-density maps at different resolution show more detail at higher resolution, (a) At low resolution (5.0 A) individual groups of atoms are not resolved, and only the rodlike feature of an

Chimeragenesis and SDM are powerful techniques that can be used to investigate the complex relationships between protein structure and function. The methods detailed here are relatively simple to perform and can be carried out in a short period of time. They are applicable to any protein type for which the cDNA is available and can be modified for many different purposes in protein engineering. 

Proteases are used in many industrial areas as well as basic research. They are classified by their mechanism of catalysis. Proteases are used in the pharmacological, food and other consumer industries to convert raw materials into a final product or to alter properties of the raw material. In biomedical research, proteases are used to study the structure of other proteins and for nthesis of peptides. The choice of a protease for an application depends in part on its specificity for peptide bonds, activity and stability. Technical advances in protein engineering have enabled alteration of these properties and allowed proteases to be used more effectively. Some easily obtained proteases can be modified so that they can substitute for proteases whose supply is limited. 

A.J. Link, M.L. Mock, D.A. Tirrell, Non-canonical amino acids in protein engineering, Curr. Opin. Biotechnol. 14(6) (2003) 603-609. 

Wells, J.A., Powers, D.B., Bott, R.R., Katz, B.A., Ultseh, M.H., Kossiakof, A.A. etal. (1987) Protein Engineering of Snbtilisin. In Protein Engineering, edited by Oxender and Fox. AlanR.Liss. 

Hahn U, Heinemann U (1994) Structure determination, modeling and site-directed mutagenesis. In Wrede P, Schneider G (eds) Concepts in protein engineering and design. De Gruyter, Berlin, p 109... 

RD Chen, A Greer, AD Dean. Structural constraints in protein engineering the coenzyme specificity of Escherichia coli isocitrate dehydrogenase. Eur J Biochem 250 578-582, 1997. 

In this study we combined two strategies common in protein engineering. Substitutions based on rational design within the nucleotide-binding pocket were used to convert E. coli IDH coenzyme specificity from NADP to NAD, while substitutions improving overall performance were identified by partial random mutagenesis at sites outside the nucleotide-binding pocket [5,12],... 

The seeds of squash plants are rich in a family of trypsin and chymotrypsin inhibitors that are approximately 35 amino acids in size and have been extensively investigated not only for their enzyme inhibitory activity, but also because they are very stable mini-protein scaffolds with applications in protein engineering. The best studied examples are Ecballium elaterium trypsin inhibitor (EETI-II) and Cucurbita maxima trypsin inhibitor (CMTI). Both X-ray and NMR have been used to characterise their structures, which incorporate a cystine-knot motif formed by three conserved disulphide bonds.93 We will describe this motif in more detail in a later section describing the plant cyclotides. 

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