Site-Directed Mutagenesis and Its Application to Protein Folding

Ep, Garvey; Cr, Matthews

doi:10.1016/b978-0-409-90116-0.50011-x

Cited by 6 publications

(4 citation statements)

References 55 publications

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“…In the last two decade, this research field has been widely investigated and described in the literature. [1][2][3] Novel proteins can also be generated via combinatorial engineering applied on a protein scaffold, which is identified as the protein portion able to retain the conformational stability when other regions are subjected to single/ multiple substitutions or peptide-segment insertions. The modifications of the protein amino acid sequence, aimed to obtain different functions without relevant effects on the protein architecture, are generally performed at the level of exposed regions, as loops.…”

Section: Introductionmentioning

confidence: 99%

Wheat Subtilisin/Chymotrypsin Inhibitor (WSCI) as a scaffold for novel serine protease inhibitors with a given specificity

et al. 2012

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WSCI (Wheat Subtilisin/Chymotrypsin Inhibitor) is a small protein belonging to the Potato inhibitor I family exhibiting a high content of essential amino acid. In addition to bacterial subtilisins and mammalian chymotrypsins, WSCI inhibits chymotrypsin-like activities isolated from digestive traits of a number of insect larvae. In vivo, as suggested for many plant proteinase inhibitors, WSCI seems to play a role of natural defence against attacks of pests and pathogens. The functional region of WSCI, containing the inhibitor reactive site (Met48-Glu49), corresponds to an extended flexible loop (Val42-Asp53) whose architecture is somehow stabilized by a number of secondary interactions established with a small β-sheet located underneath. The aim of this study was to employ a WSCI molecule as a stable scaffold to obtain recombinant inhibitors with new acquired anti-proteinase activity or, alternatively, inactive WSCI variants. A gene sequence coding for the native WSCI, along with genes coding for muteins with different specficities, could be exploited to obtain transformed non-food use plants with improved insect resistance. On the other hand, the genetic transformation of cereal plants over-expressing inactive WSCI muteins could represent a possible strategy to improve the nutritional quality of cereal-based foods, without risk of interference with human or animal digestive enzymes. Here, we described the characterization of four muteins containing single/multiple amino acid substitutions at the WSCI reactive site and/or at its proximity. Modalities of interaction of these muteins with proteinases (subtilisin, trypsin and chymotrypsin) were investigated by time course hydrolysis and molecular simulations studies.

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Section: Introductionmentioning

confidence: 99%

Wheat Subtilisin/Chymotrypsin Inhibitor (WSCI) as a scaffold for novel serine protease inhibitors with a given specificity

et al. 2012

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“…There are a number of experimental approaches designed for this purpose. The basic principle involves the use of synthetic oligonucleotides (oligonucleotide-directed mutagenesis) that are complementary to the cloned gene of interest but contain a single (or sometimes multiple) mismatched base(s) (Balland et al 1985, Garvey and Matthews 1990, Wagner and Benkovic 1990. The cloned gene is either carried by a single-stranded vector (M13 oligonucleotide-directed mutagenesis) or a plasmid that is later denatured by alkali (plasmid DNA oligonucleotide-directed mutagenesis) or heat (PCR-amplified oligonucleotide-directed mutagenesis) in order for the mismatched oligonucleotide to anneal.…”

Section: Rational Enzyme Designmentioning

confidence: 99%

Biocatalysis, Enzyme Engineering and Biotechnology

Kotzia

Platis

Axarli

et al. 2012

Food Biochemistry and Food Processing

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Enzymes are biocatalysts evolved in nature to achieve the speed and coordination of nearly all the chemical reactions that define cellular metabolism necessary to develop and maintain life. The application of biocatalysis is growing rapidly, since enzymes offer potential for many exciting applications in industry. The advent of whole genome sequencing projects enabled new approaches for biocatalyst development, based on specialised methods for enzyme heterologous expression and engineering. The engineering of enzymes with altered activity, specificity and stability, using sitedirected mutagenesis and directed evolution techniques are now well established. Over the last decade, enzyme immobilisation has become important in industry. New methods and techniques for enzyme immobilisation allow for the reuse of the catalysts and the development of efficient biotechnological processes. This chapter reviews advances in enzyme technology as well as in the techniques and strategies used for enzyme production, engineering and immobilisation and discuss their advantages and disadvantages. HO CH 2 COOH CH NH 2 Negatively charged amino acids Glutamate Glu (E) CH 2 CH 2 HOOC COOH CH NH 2 Aspartate Asn (N) CH 2 COOH HOOC CH NH 2 Positively charged amino acids Arganine Arg (R) CH 2 CH 2 CH 2 HN COOH CH NH 2 C NH NH 2 Lysine Lys (K) H 2 N COOH (CH 2) 4 CH NH 2 Histidine His (H)

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“…In the late 1980s, protein molecules were altered by site‐directed or site‐specific mutagenesis of their genes. This opened an era of protein engineering (Balland et al, 1985; Garvey and Matthews, 1990; Wagner and Benkovic, 1990). This sequence‐based design of biomolecules can be achieved after computer‐graphic modeling of individual changes.…”

Section: Key Technologies Employed In Biomolecular Engineeringmentioning

confidence: 99%

Recent Progress in Biomolecular Engineering

Ryu

Nam

2000

Biotechnology Progress

115

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During the next decade or so, there will be significant and impressive advances in biomolecular engineering, especially in our understanding of the biological roles of various biomolecules inside the cell. The advances in high throughput screening technology for discovery of target molecules and the accumulation of functional genomics and proteomics data at accelerating rates will enable us to design and discover novel biomolecules and proteins on a rational basis in diverse areas of pharmaceutical, agricultural, industrial, and environmental applications. As an applied molecular evolution technology, DNA shuffling will play a key role in biomolecular engineering. In contrast to the point mutation techniques, DNA shuffling exchanges large functional domains of sequences to search for the best candidate molecule, thus mimicking and accelerating the process of sexual recombination in the evolution of life. The phage-display system of combinatorial peptide libraries will be extensively exploited to design and create many novel proteins, as a result of the relative ease of screening and identifying desirable proteins. Even though this system has so far been employed mainly in screening the combinatorial antibody libraries, its application will be extended further into the science of protein-receptor or protein-ligand interactions. The bioinformatics for genome and proteome analyses will contribute substantially toward ever more accelerated advances in the pharmaceutical industry. Biomolecular engineering will no doubt become one of the most important scientific disciplines, because it will enable systematic and comprehensive analyses of gene expression patterns in both normal and diseased cells, as well as the discovery of many new high-value molecules. When the functional genomics database, EST and SAGE techniques, microarray technique, and proteome analysis by 2-dimensional gel electrophoresis or capillary electrophoresis in combination with mass spectrometer are all put to good use, biomolecular engineering research will yield new drug discoveries, improved therapies, and significantly improved or new bioprocess technology. With the advances in biomolecular engineering, the rate of finding new high-value peptides or proteins, including antibodies, vaccines, enzymes, and therapeutic peptides, will continue to accelerate. The targets for the rational design of biomolecules will be broad, diverse, and complex, but many application goals can be achieved through the expansion of knowledge based on biomolecules and their roles and functions in cells and tissues. Some engineered biomolecules, including humanized Mab's, have already entered the clinical trials for therapeutic uses. Early results of the trials and their efficacy are positive and encouraging. Among them, Herceptin, a humanized Mab for breast cancer treatment, became the first drug designed by a biomolecular engineering approach and was approved by the FDA. Soon, new therapeutic drugs and high-value biomolecules will be designed and produced by biomolecular ...

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Site-Directed Mutagenesis and Its Application to Protein Folding

Cited by 6 publications

References 55 publications

Wheat Subtilisin/Chymotrypsin Inhibitor (WSCI) as a scaffold for novel serine protease inhibitors with a given specificity

Wheat Subtilisin/Chymotrypsin Inhibitor (WSCI) as a scaffold for novel serine protease inhibitors with a given specificity

Biocatalysis, Enzyme Engineering and Biotechnology

Recent Progress in Biomolecular Engineering

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