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At least 19 recordsLinked to original sources
PubMed2026

Engineering Protein Stability with Small Molecules: A Review of the ecDHFR Destabilizing Domain System.

The E. coli dihydrofolate reductase (ecDHFR) destabilizing domain (DD) is a versatile post-translational tool for the conditional control of protein stability via ligand-induced stabilization. In this system, a DD-tagged protein is rapidly degraded by the proteasome unless stabilized by the antibiotic trimethoprim (TMP), allowing for conditional control of protein abundance. The ecDHFR-DD system has been successfully applied across diverse biological systems, including yeast, invertebrate models such as Drosophila, and mammalian cells, to study a broad spectrum of cellular and developmental processes. Compared with DNA- and RNA-based regulatory approaches, post-translational systems offer faster response times and more precise control, making them valuable for processes that require tight, reversible regulation. In this review, we synthesize current knowledge on the mechanisms, performance, and optimization of the ecDHFR-DD system across organisms and evaluate its advantages and limitations relative to most conditional gene expression systems. We also highlight emerging opportunities for applying the system across diverse areas, ranging from functional genomics and synthetic biology to biomedical research. Additionally, we discuss its potential application in applied biological systems, such as pest and vector management, positioning the ecDHFR-DD system as a broadly applicable platform for the precise and tunable control of protein function across diverse disciplines.

Tetrahydrofolate Dehydrogenase↗
PubMed1991

Engineering proteins to enhance their partition coefficients in aqueous two-phase systems.

We describe a novel method to partition recombinant proteins into the polymer-rich top phase in poly(ethylene glycol) (PEG)4000/potassium phosphate aqueous two-phase systems. The concept is based on fusion of a gene fragment encoding a short peptide sequence to the product gene of interest thereby changing the partitioning properties of the expressed product protein as a fusion to the peptide. The model protein in this study, ZZ, is a two domain molecule based on staphylococcal protein A (SPA) which distributes evenly in PEG/salt systems. A tetrapeptide sequence, AlaTrpTrpPro (designated the partitioning peptide), was designed by molecular modeling techniques to include exposed tryptophan residues and to have a coding DNA sequence which is possible to polymerize in an obligate head-to-tail fashion at the DNA level. Gene fragments encoding one and three partitioning peptides, respectively, were fused to the 3' end of the ZZ gene and the fusion proteins were produced intracellularly in Escherichia coli. The partition coefficients of ZZ proteins containing zero, one and three fused partitioning peptides were determined in three PEG 4000/potassium phosphate aqueous two-phase systems of different compositions. In all three phase systems, there were dramatic effects on the partition coefficient by the fused partitioning peptides. In the phase system with the largest effects, the partition coefficient was enhanced from 1.6 to 11.6 by fusing one tetrapeptide sequence to the 147 amino acid model ZZ protein. By the fusion of three partitioning peptides, the coefficient was increased to 96.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Engineering proteins for purification.

Over the past decade, a new protein purification technique has emerged as a result of recombinant DNA technology. DNA, encoding additional polypeptide or protein tags, is fused to the gene of interest. Expression of these gene fusions results in protein fusions which may be purified by techniques using the properties of the additional polypeptide tag. This has eliminated the need for extensive screening and optimization procedures previously required for purification.

Chromatography, Affinity↗
PubMed1992

New recombinant DNA methodology for protein engineering.

Over the past year considerable progress has been made in the application of recombinant DNA technology to protein engineering. A number of new methods for gene synthesis and mutagenesis have been reported that simplify the construction of novel coding sequences. The polymerase chain reaction plays an increasingly important role in these methods. Amino acid diversity has been extended by the incorporation of unnatural amino acids via coupled in vitro transcription-translation methods. Novel random mutagenesis strategies have been developed that substitute amino acids with a desired chemical character at a given position, thereby generating a sophisticated library of protein variants.

Amino Acids↗
PubMed1992

Structural and functional properties of hen egg-white lysozyme deamidated by protein engineering.

The structural and functional properties of lysozymes genetically deamidated at positions 103 (N103D) and 106 (N106D) were studied by a protein engineering technique. The wild-type and mutant lysozymes were expressed in Saccharomyces cerevisiae and purified from the cultivation medium in two steps by cation-exchange chromatography on CM-Toyopearl. The lytic activity of deamidated lysozymes was almost the same as that of wild lysozyme, although the optimal pH of activity was slightly shifted to lower pH by the deamidation. The Gibbs free energy changes of unfolding (delta G) at 20 degrees C for N103D and N106D were almost the same as that of wild-type. On the other hand, the structural flexibility of lysozymes, estimated by protease digestion, was significantly increased by the deamidation. The surface functional properties of deamidated lysozymes were considerably enhanced, compared to those of wild-type lysozyme. These results suggest that structural flexibility is an important governing factor in surface functional properties of proteins, regardless of their structural stability.

Animals↗
PubMed1992

Protein engineering for molecular electronics.

Recombinant DNA technology allows the manipulation of the physical properties of proteins that perform electron transport and photochemical processes. Recent work is reviewed that has a potential impact on the development of molecular electronic devices, within a general framework outlining strategies for device fabrication. This review is also published in Current Opinion in Structural Biology 1992, 2:587-592.

Bacteriorhodopsins↗
PubMed1990

Protein engineering for thermostability.

Studies with small, monomeric proteins indicate that, to some extent, the effects of amino acid substitutions can be predicted. However, conformational and other changes may complicate the prediction. Site-directed mutagenesis is leading both to a better understanding of protein stability and to the production of more stable proteins.

Amino Acid Sequence↗
PubMed1992

The organisation of production of genetically-engineered proteins in yeast.

The use of a variety of genetic-engineering techniques to introduce foreign DNA into living yeast cells has resulted in the production in these cells of the corresponding proteins. These include enzymes, antibodies, vaccines, anti-viral agents, and hormones. This article discusses the techniques of genetic engineering in yeast and suggests the lines of future progress in the economic production of novel proteins for use in therapy.

Plasmids↗
PubMed1991

New molecular biology methods for protein engineering.

This review outlines recent advances in the application of molecular biological techniques to the study of protein structure and function. The chapter is divided into four main sections: methods for oligonucleotide-directed mutagenesis; mutational strategies for identifying functional residues and domains; systems for expression; and future developments. Few new methods were reported in 1990; however, a number of the papers that appeared represent refinements of previously reported strategies. This review is also published in Current Opinion in Structural Biology 1991, 1:605-610.

Biotechnology↗