生物化学技术

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 ...

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 te ...

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 s ...

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 mate ...

In nature, enzymes often form multienzyme complexes to enhance their catalytic efficiencies and, �moreover, evolve into genetically fused multidomain enzymes. Inspired by a natural fusion cytochrome P450 (P450) containing a monooxygenase domain and a reductase domain, we have de ...

In this chapter, we describe a method to identify amino acids (AA) by capillary electrophoresis in conjunction with light-emitting diode-induced fluorescence (LEDIF). First, amino acids labeled with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide are conver ...

Methods for the cloning of large numbers of open reading frames (ORFs) into expression vectors are of critical importance for diverse disciplines in biology. Here I describe a system termed FX cloning that facilitates the high-throughput generation of expression constructs. FX cloning ...

We report a capillary electrophoresis method by field-amplified sample injection (FASI) for the separation and detection of free plasma arginine and dimethylated arginines. Protein elimination from plasma was performed by acetonitrile/ammonia precipitation. Supernat ...

Biomarker analysis is pivotal for disease diagnosis and one important class of biomarkers is constituted by proteins and peptides. This review focuses on protein and peptide analyses from biological fluids performed by capillary electrophoresis. The various strategies that have ...

The analysis of proteins and peptides in biological fluids is becoming more important as they are potential sources of diagnostic biomarkers of disease. The complexity of body fluids is such that no single technique can both identify and quantify all the constituents present. It therefore r ...

In this chapter, the potential of capillary electrophoresis-mass spectrometry (CE-MS) for peptide analysis is demonstrated by the presentation of two different strategies typically followed in analysis of these biomolecules by CE-MS. The first one is a target-based approach and it is ...

Here, we describe a capillary electrophoresis method for evaluating proteolysis of the amyloid beta peptide (Aβ) 1–40. This method is suitable for kinetic studies, demands little specialized equipment, and consumes only small quantities of a commercially available substrate whose ...

Glutelin, the major seed storage protein of rice (Oryza sativa L.), consists of multiple polymeric and monomeric subunits. Each subunit is composed of an α and a β polypeptide that are covalently linked by a disulfide bond. To analyze the microheterogeneous glutelin subunits using capillary e ...

This chapter describes a complete procedure for obtaining protein fingerprints of microorganisms using capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). Staphylococcus aureus, a human pathogen responsible of frequent and resistant inf ...

Hemoglobinopathies are genetic disorders of globin chains characterized by the decreased expression of α- or β-globin chains (thalassemias) or by the synthesis of an abnormal protein (hemoglobin variants in, e.g., sickle cell disease). The screening of most hemoglobinopathies rel ...

Sample preparation and laser-induced fluorescence detection are two key steps of the analytical methodology to determine by capillary electrophoresis low concentrations of proteins in complex sample matrices. In this chapter the options of performing both steps in different wa ...

Capillary electrophoresis (CE) is an interesting technique for protein and peptide analysis. However, one of the major problems concerns sample adsorption on the internal capillary wall. The use of non-covalent coatings using highly charged polyelectrolytes is an efficient, simp ...

Due to the inherent disadvantage of biomarker dilution in complex biological fluids such as serum/plasma, urine, and saliva, investigative studies directed at tissues obtained from the primary site of pathology probably afford the best opportunity for the discovery of disease bioma ...

Enzymes are an important class of drug targets. In the early stage of the drug discovery, the major task is to find out the inhibitors of a given enzyme target in a compound library. Herein we describe a method for screening the enzyme inhibitors in the complex mixtures (such as the natural extracts) by capil ...

Carboxylesterases are important enzymes for xenobiotic metabolism and are receiving increasing attention in the context of cancer therapies. Quantification of individual carboxylesterase activity is important since protein levels do not always correlate to activity and ...