Engineering Protein Sequences

One of the major applications of recombinant DNA technology has been to produce large amounts of commercially relevant proteins, including enzymes, receptors, and peptide messengers of various sorts. The sequences of these proteins, at least in the initial stages of investigation and production, have been those found in nature, so that the structure and function of the protein products of cloning would be the same as those of natural proteins extracted from tissue, serum, and so forth. 

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Besides sensitive methods for the analysis of proteins, bioinformatics is one of the key components of proteome research. This includes software to monitor and quantify the separation of complex samples, e.g., to analyze 2DE images. Web-based database search engines are available to compare experimentally measured peptide masses or sequence ions of protein digests with theoretical values of peptides derived from protein sequences. Websites for database searching with mass spectrometric data may be found at http //www.expasy.ch/tools, http //prospector.ucsf. edu/ and http //www.matrixscience.com. 

Since most synthetic applications require enzymes catalyzing nonnatural substrates, their properties often have to be improved. One way to achieve this is to optimize reaction conditions such as pH, temperature, solvents, additives, etc. [6-9]. Another way is to modulate the substrates without compromising the synthetic efficiency of the overall reaction [10]. In most cases for commercial manufacturing, however, the protein sequences have to be altered to enhance reactivity, stereoselectivity and stability. It was estimated that over 30 commercial enzymes worldwide have been engineered for industrial applications [11]. Precise prediction of which amino acids to mutate is difficult to achieve. Since the mid 1990s, directed evolution... 

Half-lives were measured in yeast for the j8-galactosidase protein modified so that in each experiment it had a different amino-terminal residue. (See Chapter 9 for a discussion of techniques used to engineer proteins with altered amino acid sequences.) Half-lives may vary for different proteins and in different organisms, but this general pattern appears to hold for all organisms. 

Even if we restrict our design to a small number of sites in the protein, the combinatorial possibilities quickly approach astronomical dimensions. If we consider mutations at 10 sites and a subset of 10 amino acids, we have 1010 possible variants. Although experimental approaches are under development that can actually search large subsets of protein sequence space, it is not at all a small feat to identify those variants that give rise to a stable structure and at the same time come close to the desired features. Therefore, computational approaches that, with some reliability, are able to pick those variants having a stable structure are desirable instruments in the protein engineer s toolbox. 

The NCBI s Entrez is apowerful database search engine. This integrated search engine provides a menu (http //www.ncbi.nih.gov/Entrez/index.html) offering the user selections to search biomedical literature (PubMed), databases on OMIM, nucleotide sequence, protein sequence, whole genome sequences, 3D macromolecular structures, taxonomy (organisms in GenBank), SNP,... 

Gavel Y, von Fleijne G (1990), Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites implications for protein engineering, Protein Eng. 3 433-442. 

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