Purification from bacteria

Chirazymes. These are commercially available enzymes e.g. lipases, esterases, that can be used for the preparation of a variety of optically active carboxylic acids, alcohols and amines. They can cause regio and stereospecific hydrolysis and do not require cofactors. Some can be used also for esterification or transesterification in neat organic solvents. The proteases, amidases and oxidases are obtained from bacteria or fungi, whereas esterases are from pig liver and thermophilic bacteria. For preparative work the enzymes are covalently bound to a carrier and do not therefore contaminate the reaction products. Chirazymes are available form Roche Molecular Biochemicals and are used without further purification. 

The individual steps in the elongation of the fatty acid chain are quite similar in bacteria, fungi, plants, and animals. The ease of purification of the separate enzymes from bacteria and plants made it possible in the beginning to sort out each step in the pathway, and then by extension to see the pattern of biosynthesis in animals. The reactions are summarized in Figure 25.7. The elongation reactions begin with the formation of acetyl-ACP and malonyl-ACP, which... 

As noted above, whole-cell MALDI-TOF MS was intended for rapid taxonomic identification of bacteria. Neither the analysis of specific targeted bacterial proteins, nor the discovery of new proteins, was envisioned as a routine application for which whole cells would be used. An unknown or target protein might not have the abundance or proton affinity to facilitate its detection from such a complex mixture containing literally thousands of other proteins. Thus, for many applications, the analysis of proteins from chromatographically separated fractions remains a more productive approach. From a historical perspective, whole-cell MALDI is a logical extension of MALDI analysis of isolated cellular proteins. After all, purified proteins can be obtained from bacteria after different levels of purification. Differences in method often reflect how much purification is done prior to analysis. With whole-cell MALDI the answer is literally none. Some methods attempt to combine the benefits of the rapid whole cell approach with a minimal level of sample preparation, often based on the analysis of crude fractions rather... 

Post-translational modifications, such as phosphorylation, complex glycosylation, and lipidation, typically occur in eukaryotic organisms. Therefore, their expression in prokaryotic systems like Escherichia coli is difficult. However, it should be noted that via clever engineering and coexpression of specific enzymes, access can be granted to specific lipidated proteins via expression in bacteria, for example, via the expression of A -myristoyltransferase in E. coli Eukaryotic systems that can be used for the expression of post-translationally modified proteins are yeast and Dictyostelium discoidum. Furthermore, lipidated proteins, such as the Rah proteins, can be obtained via purification from tissue sources or from membrane fractions of insect cells that had been infected with baculovirus bearing a Rah gene. ... 

Braun, P, Hu, Y., Shen, B., HaUeck, A., Koundinya, M., Harlow, E. and LaBaer, J. (2002). Proteome-scale purification of human proteins from bacteria. Proc. Natl. Acad. Sci. USA 99, 2654-2659. 

Before attempting to purify a protein, the first thing to consider is the source of starting material. Proteins differ in their cellular and tissue distribution, and thus if a protein is known to be abundant in one particular tissue (e.g. kidney) it makes sense to start the purification from this source. Also, some sources are more readily available than others and this should be taken into account too. Nowadays, with the use of recombinant DNA techniques (see Topics II and 16), even scarce proteins can be expressed in bacteria or eukaryotic cells and relatively large amounts of the protein subsequently obtained. 

One method that has been used to acquire carbohydrates is isolation and purification from natural sources such as human or animal tissue, milk, urine, plants, and bacteria (see cross reference Isolation of glycans). Access to homogeneous carbohydrate stmctures can be challenging due to the difficulties in separation of complex mixtures, identification of carbohy-drate(s) contained within each fraction, and preparation of sufficient quantities from the limited amounts present in a particular sample. Alternatively, mixtures of unknown composition can be used to survey a broad repertoire of the glycome. On identification of a mixture containing one or more members with interesting receptor-binding properties, the mixture can then be deconvoluted by further fractionation and separation by routine analytical techniques (13). 

Heparin/heparan, hyaluronan, and chondroitin are three prevalent glycosaminoglycans. Vertebrates use glycosaminoglycans in structural, recognition, adhesion, and signaling roles. Chemical synthesis of naturally occurring polysaccharides is considered to be impractical. Most polysaccharides, especially those from bacteria origins, are obtained by purification from natural sources or from cell culture, enzymatic approaches have been increasingly applied to obtain some structures. 

The subsequent purification of other ion pumps has revealed a large family of evolutionarily related ion pumps including proteins from bacteria, archaea, and all eukaryotes. These pumps are specific for an array of ions. Of particular interest are the Ca2+ ATPase, the enzyme that transports Ca2+ out of the cytoplasm and into the sarcoplasmic reticulum of muscle cells, and the gastric H+-K+ ATPase, the enzyme responsible for pumping sufficient protons into the stomach to lower the pH below 1.0. These enzymes and the hundreds of known homologs, including the Na+-K+ ATPase, are referred to as P-type ATPases because they form a key phosphorylated intermediate. In the formation of this intermediate, a phosphoryl group obtained from the hydrolysis of ATP is linked to the side chain of a specific conserved... 

The subsequent purification of other ion pumps has revealed a large family of evolutionarily related ion pumps including proteins from bacteria, archaea, and all eukaryotes. These pumps are specific for an array... 

Plasmid is routinely recovered from bacteria by an alkaline lysis procedure, which lyses the bacterial cell while maintaining bacterial DNA attachment to the cell wall. This procedure enables subsequent precipitation of bacterial DNA and cellular debris, leaving a crude preparation enriched in plasmid. We routinely use a plasmid DNA purification kit provided by Qiagen that utilizes the alkaline lysis method for harvesting, and anion exchange column chromatography for rapid purification. We refer the reader to the detailed instructions provided in the kit by the manufacturer, which we have not found necessary to modify for purification of laboratory-use plasmid DNA. 

Several kits are commercially available for the extraction of RNA from plant, bacteria, or mammalian sources. We have used the RNeasy Mini Kit (Qiagen, Valencia, CA) for the extraction of RNA from bacteria following the procedure recommended by the manufacturer (92). This kit relies on guanidine thiocyanate-silica isolation of RNA. Here, guanidine thiocyanate serves as a chaotropic agent, which both lyses cells and inactivates nucleases. In its presence, the released nucleic acids bind to silica particles, which provide a solid phase from which the collected RNA can later be eluted using water (93). This kit is suitable for the isolation and purification of up to 100 pg RNA molecules >200... 

Classically, plants and bacteria have been the major sources for the purification of PLDs. The function of PLD in plants is not known although it may be involved in cell turnover and energy utilization during different cycles in plant life. Bacterial PLDs in some cases are toxins that can lead to severe cellular damage either alone or in combination with other proteins secreted from bacteria. These bacterial enzymes may also serve to help provide nutrients for the cell such as inorganic phosphate, as do the bacterial PLCs [2]. 

Part B Expression and Purification of Tagged Chemokines from Bacteria for Enzymatic Labeling... 

Purification of Recombinant Tagged Chemokines from Bacteria... 

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