生物化学技术

This chapter presents in detail the process used in high throughput bacterial production of recombinant human proteins for crystal structure determination. The core principles are: (1) Generating at least 10 truncated constructs from each target gene. (2) Ligation-independent cl ...

Most functional entities within cells are composed of protein complexes. The actual challenge for structural biologists is to purify these complexes, or at least functional subcomplexes, in sufficiently large amounts for structural characterization. One major technique for as ...

Cell-free protein synthesis offers fast and inexpensive access to selectively isotope labeled proteins thath can be measured by NMR spectroscopy in the presence of all the unlabeled proteins in the reaction mixture. No chromatographic purification is required. Using an extract from ...

Expression of insoluble protein in E. coli is a major bottleneck of high throughput structural biology projects. Refolding proteins into native conformations from inclusion bodies could significantly increase the number of protein targets that can be taken on to structural studies. ...

Approximately one third of the proteins encoded in prokaryotic and eukaryotic genomes reside in the membrane. However, membrane proteins comprise only a minute fraction of the entries in protein structural databases. This disparity is largely due to inherent difficulties in the expr ...

The systematic structural analysis of many target proteins involves generating expression clones in high throughput. This requires robust laboratory procedures and benefits from laboratory automation and data management systems. This chapter gives an overview of the Protein ...

Mass spectrometry (MS) is widely used within structural and functional proteomics for a variety of tasks including protein quality assessment, identification, and characterization. MS is used routinely for the determination of the total mass of proteins, including N-glycosylat ...

Transition metals are among the most common ligands that contribute to the biochemical and physiological properties of proteins. In the course of structural proteomic projects, the detection of transition metal cofactors prior to the determination of a high-resolution structure ...

Understanding the functions of every protein in the proteome is one of the great challenges of the postgenomic era. Global genome sequencing efforts revealed that in any genome 30–50% of genes encode proteins with unknown function (hypothetical proteins). To directly test purified hypo ...

The production of crystals suitable for high-resolution structure determination is still one of the major bottlenecks in the structure determination process. This is especially true in structural genomics (SG) consortia, where the implementation of protein-specific purifi ...

In the structural genomics period traditional methods for protein crystallization have been eclipsed by automation using batch or vapor diffusion equilibration to find conditions conducive for protein crystal growth. Although many globular and soluble proteins predomina ...

Trace fluorescent labeling, typically less than 1%, can be a powerful aid in macromolecule crystallization. Precipitation concentrates a solute, and crystals are the most densely packed solid form. The more densely packed the fluorescing material, the brighter the emission from it; thu ...

Microfluidic technologies enable a relatively new approach to macromolecular crystallization, but offer several significant advantages over more traditional techniques. Microfluidic devices provide significant savings in the amount of material required to complete a ...

The crystallization step is considered to be a major bottleneck in the process of structure determination by x-ray crystallography. Successful crystallization requires both the formation of nuclei that are capable of supporting crystal growth, and a subsequent crystal growth. Nuc ...

Most high throughput structural proteomics centers use the sitting-drop method to obtain diffracting crystals for three-dimensional (3D) structure determination of biological macromolecules by x-ray crystallography. Although several robotic systems are available f ...

Significant time and effort are often required to solve and complete a macromolecular crystal structure. The development of automated computational methods for the analysis, solution, and completion of crystallographic structures has the potential to produce minimally bias ...

In the age of structural proteomics when protein structures are targeted on a genome-wide scale, the identification of proteins that are amenable to analysis using x-ray crystallography or NMR spectroscopy is the key to high throughput structure determination. NMR screening is a benefi ...

Microcoil NMR spectroscopy is based on the increase of coil sensitivity for smaller coil diameters (approximately 1/d). Microcoil NMR probes deliver a remarkable mass-based sensitivity increase (8- to 12-fold) when compared with commonly used 5-mm NMR probes. Although microcoil NMR p ...

Cross-linking in combination with mass spectrometry can be used as a tool for structural modeling of protein complexes and multidomain proteins. Although cross-links represent only weak structural constraints, the combination of a limited set of experimental cross-links with mo ...

The initial objective of the Berkeley Structural Genomics Center was to obtain a near complete three-dimensional (3D) structural information of all soluble proteins of two minimal organisms, closely related pathogens Mycoplasma genitalium and M. pneumoniae. The former has fewer t ...