Springer protocols feed by Biochemistry 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Reconstructing Evolutionary Adaptive Paths for Protein Engineering
Reconstructing Evolutionary Adaptive Paths (REAP) is one of several methods to improve enzyme functionality. This approach incorporates computational and theoretical aspects of protein engineering to create a focused library of protein variety with a high degree of functionality. In contrast to other techniques like DNA shuffling, REAP allows a library to have diverse functionality among relatively few variants. REAP is a low-throughput method which takes advantage of natural selection and uses ancestral protein sequences to direct gene mutations, thereby creating a library with a high density of viable proteins....
Source: Springer protocols feed by Biochemistry - February 21, 2013 Category: Biochemistry Source Type: news
Residue-Specific Incorporation of Unnatural Amino Acids into Proteins In Vitro and In Vivo
The incorporation of noncanonical (unnatural) amino acids into proteins offers researchers the ability to augment the biochemical functionality of proteins for a myriad of applications including bioorthogonal conjugation, biophysical and structural studies, and the enhancement or de novo creation of novel enzymatic activities. The augmentation of a protein throughout its coding sequence by global residue-specific incorporation of unnatural amino acid analogs is an attractive technique for studying both the utility of individual chemistries available through unnatural amino acids and the general effects of unnatural amino a...
Source: Springer protocols feed by Biochemistry - February 21, 2013 Category: Biochemistry Source Type: news
In Vitro Evolution of Enzymes
In the past decade, in vitro evolution techniques have been used to improve the performance or alter the activity of a number of different enzymes and have generated enzymes de novo. In this review, we provide an overview of the available in vitro methods, their application, and some general considerations for enzyme engineering in vitro. We discuss the advantages of in vitro over in vivo approaches and focus on ribosome display, mRNA display, DNA display technologies, and in vitro compartmentalization (IVC) methods. This review aims to help researchers determine which approach is best suited for their own experimental nee...
Source: Springer protocols feed by Biochemistry - February 21, 2013 Category: Biochemistry Source Type: news
TAL Effector Nuclease (TALEN) Engineering
TALENs, fusion proteins of DNA binding domains of TAL (transcription activator-like) effectors and the DNA cleavage domains of endonuclease FokI, have emerged as genetic tools for targeted gene modification, holding great potential for basic and applied research, even for gene therapy. Here we present a simple and efficient approach to custom-engineering TALEN genes with four basic TAL repeats and their DNA recognition cipher. The “modular assembly” method also involves the “Golden Gate” cloning strategy, using 53 ready-to-use plasmids in just two rounds of restriction and ligation to assemble TALEN...
Source: Springer protocols feed by Biochemistry - February 21, 2013 Category: Biochemistry Source Type: news
Flow Cytometric Assays for Interrogating LAGLIDADG Homing Endonuclease DNA-Binding and Cleavage Properties
A fast, easy, and scalable method to assess the properties of site-specific nucleases is crucial to understanding their in cellulo behavior in genome engineering or population-level gene drive applications. Here we describe an analytical platform that enables high-throughput, semiquantitative interrogation of the DNA-binding and catalytic properties of LAGLIDADG homing endonucleases (LHEs). Using this platform, natural or engineered LHEs are expressed on the surface of Saccharomyces cerevisiae yeast where they can be rapidly evaluated against synthetic DNA target sequences using flow cytometry. (Source: Springer proto...
Source: Springer protocols feed by Biochemistry - February 21, 2013 Category: Biochemistry Source Type: news
GFP Reporter Screens for the Engineering of Amino Acid Degrading Enzymes from Libraries Expressed in Bacteria
We describe a high-throughput fluorescence activated cell sorting (FACS) assay for detecting the catalytic activity of amino acid degrading enzymes in bacteria, at the single cell level. This assay relies on coupling the synthesis of the GFP reporter to the catalytic activity of the desired amino acid degrading enzyme in an appropriate E. coli genetic background. The method described here allows facile screening of much larger libraries (106–107) than was previously possible. We demonstrate the application of this technique in the screening of libraries of bacterial and human asparaginases and also for the catalytic ...
Source: Springer protocols feed by Biochemistry - February 21, 2013 Category: Biochemistry Source Type: news
Determining Enzyme Kinetics via Isothermal Titration Calorimetry
Isothermal titration calorimetry (ITC) has emerged as a powerful tool for determining the thermodynamic properties of chemical or physical equilibria such as protein–protein, ligand–receptor, and protein–DNA binding interactions. The utility of ITC for determining kinetic information, however, has not been fully recognized. Methods for collecting and analyzing data on enzyme kinetics are discussed here. The step-by-step process of converting the raw heat output rate into the kinetic parameters of the Michaelis–Menten equation is explicitly stated. The hydrolysis of sucrose by invertase is used to de...
Source: Springer protocols feed by Biochemistry - February 21, 2013 Category: Biochemistry Source Type: news
A Tripartite Fusion System for the Selection of Protein Variants with Increased Stability In Vivo
We describe here a genetic selection system that directly links protein stability to antibiotic resistance, allowing one to directly select for mutations that stabilize proteins in vivo. Our technique is based on a tripartite fusion in which the protein to be stabilized is inserted into the middle of the reporter protein β-lactamase via a flexible linker. The gene encoding the inserted protein is then mutagenized using error-prone PCR and the resulting plasmid library plated on media supplemented with increasing concentrations of β-lactam antibiotic. Mutations that stabilize the protein of interest can easily be ...
Source: Springer protocols feed by Biochemistry - February 21, 2013 Category: Biochemistry Source Type: news
In Vitro Directed Evolution of Enzymes Expressed by E. coli in Microtiter Plates
A method is described for using 96-well plates to prepare libraries of Escherichia coli cultures for screening a library of gene variants. This approach bypasses colony-picking to allow standard molecular biology laboratories to carry out directed evolution efficiently with a 96-well plate-reader and multichannel pipettes. Initial screens are applied to cultures that are rapidly prepared by diluting transformed cells so that an average of four cells starts each culture. Subsequent screens are used to isolate individual enzyme-expressing clones that exhibit activity higher than the parental clone. The outlined method also i...
Source: Springer protocols feed by Biochemistry - February 21, 2013 Category: Biochemistry Source Type: news
Screening Libraries for Improved Solubility: Using E. coli Dihydrofolate Reductase as a Reporter
Low protein solubility is a problem in many areas of protein science. Although chemical methods have been developed to solubilize proteins these are not always effective and add to the cost of producing the protein. One way of overcoming these difficulties is to evolve the protein to be more soluble. A major hurdle in this process is the ability to select mutant proteins with enhanced solubility from a large library of randomly mutated proteins. In this article, we describe such a method. The method relies on the fact that increasing the expression of dihydrofolate reductase (DHFR) makes Escherichia coli resistant to Trime...
Source: Springer protocols feed by Biochemistry - February 21, 2013 Category: Biochemistry Source Type: news
Rational Protein Sequence Diversification by Multi-Codon Scanning Mutagenesis
A new method for protein sequence diversification is based on generating random codon mutations to an encoding DNA. This allows for the scanning of user-defined amino acid changes to any protein of interest, and is an alternative to traditional directed evolution strategies. This chapter describes the procedures required to apply this technology to any protein of interest. The resulting libraries can then be screened for new or improved protein function. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - February 21, 2013 Category: Biochemistry Source Type: news
Promiscuity-Based Enzyme Selection for Rational Directed Evolution Experiments
Error-prone PCR, DNA shuffling, and saturation mutagenesis are techniques used by protein engineers to mimic the natural “evolutionary walk” that conjures new enzymes. Rational design is often critical in efforts to accelerate this “random walk” into a “resolute sprint.” Previous work by our group established a computational method for detecting active sites (CLASP) based on spatial and electrostatic properties of catalytic residues, and a method to quantify promiscuous activities in a wide range of proteins (PROMISE). Here, we describe a rational design flow (DECAAF) based on the PROMIS...
Source: Springer protocols feed by Biochemistry - February 21, 2013 Category: Biochemistry Source Type: news
Gene Assembly and Combinatorial Libraries in S. cerevisiae via Reiterative Recombination
While mutagenesis of single genes is now common practice in molecular biology, engineering multiple target genes still requires complex cloning techniques and thus is limited to expert laboratories. Here, we describe “Reiterative Recombination,” a user-friendly DNA assembly technique in Saccharomyces cerevisiae for the integration of an indefinite number of DNA fragments sequentially into the yeast genome. The high efficiency of chromosomal integration can further be utilized for the assembly of large combinatorial libraries for metabolic engineering. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - February 21, 2013 Category: Biochemistry Source Type: news
Protein Engineering: Single or Multiple Site-Directed Mutagenesis
Site-directed mutagenesis techniques are invaluable tools in molecular biology to study the structural and functional properties of a protein. To expedite the time required and simplify methods for mutagenesis, we recommend two protocols in this chapter. The first method for single site-directed mutagenesis, which includes point mutations, insertions, or deletions, can be achieved by an inverse PCR strategy with mutagenic primers and the high-fidelity Phusion® DNA Polymerase to introduce a site-directed mutation with exceptional efficiency. The second method is for engineering multiple mutations into a gene of interest...
Source: Springer protocols feed by Biochemistry - February 21, 2013 Category: Biochemistry Source Type: news
Gene Synthesis by Assembly of Deoxyuridine-Containing Oligonucleotides
Gene synthesis is an invaluable technique in synthetic and molecular biology for synthesis of artificial genes, operons, and even genomes. In many cases the traditional methods for obtaining functional DNA sequences through cloning are not applicable due to the novelty of genetic material. Here, we describe the simple and economical DNA synthesis method based on USER™ technology. The method consists of (1) synthesis of building blocks up to 500 bp; (2) assembly of genes up to 3 kb; (3) error correction reassembly; and (4) assembly of operons up to 15 kb if needed. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - February 21, 2013 Category: Biochemistry Source Type: news
