Directed biological synthesis of proteins

The biological synthesis of proteins is central to so much chemical biology research today. Modern day biological synthesis of proteins requires that all proteins are purified from one organism or another. If particularly large quantities of proteins (mg-g levels) are required then recombinant techniques and the growth of recombinant factory organisms are often 

Immobilised metal ions may be used to specifically coordinate with proteins so that they maybe specifically isolated. The most common use of IMAC is that the protein is engineered to carry a sequence cluster of four to eight histidine residues, enabling simple purification. 

This tag is based on the natural maltose binding protein (MBP) from E. coli. The protein of interest may be fused to the maltose binding protein, a tag that enables purification by affinity to immobilised amylose. Maltose may then be used to elute the protein, Unlike GST, MBP is monomeric, but it is one of the largest tags (42 kDa). MBP is one of the best tags for encouraging solubilisation of an insoluble or sparingly soluble protein of interest. 

The real beauty of this system is that the self-cleavage reaction can be performed on an affinity column directly after purification on cellulose beads, so that the protein of interest can be eluted directly without any attached protein affinity tags. Naturally, fusion proteins may be engineered with an intein protein affinity tag fused at either the N- or C-terminus of the protein of interest, as appropriate. 

6 Other affinity tags and radio-labelling of proteins 

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Figure 1 The central dogma of molecular biology. The central dogma describes the flow of information between the principle biopolymers of the cell. The primary exchange is shown horizontally, left to right, namely, DNA directs the synthesis of RNA, which in turn directs the synthesis of protein. Alternate observed exchanges are depicted off the horizontal with less prominent lines. The letters "P" and "R" indicate that protein or RNA, respectively, are required for particular exchanges.

DNA and RNA molecnles direct the synthesis of proteins in the ceU, a subject that is beyond the scope of this book. Introductory texts in biochemistry and molecular biology explain this process. 

S-Adenosyl-L-methionine is the important methyl donor in biological transmethylation to form S-adenosyl-L-homocysteine, which is hydrolyzed to adenosine and homocysteine by S-adenosyl-L-homocysteine hydrolase (E.C. 3.3.1.1) in vivo. However, equilibrium of the S-adenosyl-L-homocysteine hydrolase reaction favors the direction toward synthesis of S-adenosyl-L-homocysteine. Shimizu et al. developed a simple and efficient method for the high yield preparation of S-adenosyl-L-homocysteine with S-adenosyl-L-homocysteine hydrolase of Alcaligenes faecalis, in which the cellular content of S-adenosyl-L-homocysteine hydrolase was about 2.5% of the total soluble protein. S-Adenosyl-r-homocysteine was produced at a concentration of about 80 g I. 1 with a yield of nearly 100% 661. However, when racemic... 

RNA is a special macromolecule in biology because it is able to store and transmit information as well as catalyze some processes. One of these processes is protein synthesis where mRNA molecules direct the assembly of proteins on ribosomes. In contrast to DNA, it has a single strand structure. There are several types of RNA, which are differentiated by their functions. The three most common types are messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA). 

The power of the pooled GST fusion protein approach will increase as new biochemical reagents and assays become available. The development of chemical probes for biological processes, termed chemical biology, is a rapidly advancing field. For example, the chemical synthesis of an active site directed probe for identification of members of the serine hydrolase enzyme family has recently been described (Liu et al., 1999). The activity of the probe is based on the potent and irreversible inhibition of serine hydrolases by fluorophosphate (FP) derivatives such as diisopropyl fluorophosphate. The probe consists of a biotinylated long-chain fluorophosphonate, called FP-biotin (Liu et al., 1999). The FP-biotin was tested on crude tissue extracts from various organs of the rat. These experiments showed that the reagent can react with numerous serine hydrolases in crude extracts and can detect enzymes at subnanomolar... 

Prize in 1963 for inventing a new general method to synthesize important polymers, a method that uncovered much new basic science. A Nobel Prize in 1984 went to Robert Bruce Merrifield for his invention of a general approach to the synthesis of polypeptides and proteins, in a style directly reminiscent of the biological method used in such synthesis. 

In contrast to the biopharmaceuticals discussed thus far (recombinant proteins and gene therapy products), antisense oligonucleotides are manufactured by direct chemical synthesis. Organic synthetic pathways have been developed, optimized and commercialized for some time, as oligonucleotides are widely used reagents in molecular biology. They are required as primers, probes and for the purposes of site-directed mutagenesis. 

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