Protein engineering substitutions

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],... 

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. 

As noted earlier, genetic engineering methods can be used not only to make a particular protein but also to introduce amino acid substitutions into the protein in a controlled fashion. These protein engineering methods can be used to modify proteins to facilitate their purification. 

Other, more subtle applications of protein engineering to enhance the isolation of a particular protein involve amino acid substitutions, carefully f hosen to avoid interference with the protein s activity, which confer special binding or partitioning properties on that protein [22]. Chemical engineers, armed with awareness of the types of interactions possible and the options and overall goals of separation processes, are well equipped to attack these protein design problems and should be increasingly involved in this area in the future. 

E. coli class I RNR was evident already in 1983, long before the three-dimensional structures were known or protein engineering studies had been adopted. A common net result of ineubations with 2 -substituted substrate analogues is inactivation of the R2 eomponent by loss of the tyrosyl radical, and formation of a transient radieal loealised to the active site of the enzyme which eventually leads to inaetivation of protein R1 by covalent modification. This effective dual inhibition of RNR by some of the 2 -substituted substrate analogues is eurrently explored in antiproliferative treatment in clinical trials (Nocentini, 1996). 

Sutcliffe, M. J., Hayes, F. R. F., Blundell, T. L. Knowledge-based modelling of homologous proteins, part II Rules for the conformations of substituted sidechains. Protein Engineering, 1987, 1,385-392. 

It is quite clear that protein engineering will contribute substantially to future investigations of electron transfer in flavocytochrome 62. To date protein engineering has been used to generate a number of single amino acid substitutions and has allowed the independent expression of the two functionally distinct domains of the enzyme. These two approaches can be readily combined, for example, to express the flavodehydrogenase domain with an active site mutation, thereby sim-plyfing analysis of electron transfer to FMN without interference from the cytochrome domain. 

There are several examples in which protein engineering has been used to introduce specific functionalities at different points in a protein via site-directed mutagenesis, to facilitate the synthesis of peptide polymer hybrid materials. Stayton et al. [91] used protein engineering to create an N49C mutant streptavidin, in which the asparagine at position 49 was substituted... 

Benner, S. A., Cohen, M. A., Gonnet, G. H. (1994) Amino acid substitution during functionally constrained divergent evolution of protein sequences, Protein Engineering 7 1323-1332. 

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