Proteolytic digestion products

The product of the pol gene (reverse transcriptase) is translated as a larger polyprotein, on the same mRNA that is used for the gag protein alone (see Fig. 26-30). The polyprotein, or gag-pol protein, is then trimmed to the mature reverse transcriptase by proteolytic digestion. Production of the polyprotein requires a translational frameshift in the overlap region to allow the ribosome to bypass the UAG termination codon at the end of the gag gene (shaded pink in Fig. 1). 

Since gel permeation discrimination depends on Re, it is apparent that dramatically enhanced resolution is obtainable in 6M GuHCl. This factor has led to the use of this technique for analysis of such complex mixtures as proteolytic digestion products (12,13) and red cell membrane proteins (14). An added dividend of the method is recovery of the isolated polypeptide components for further physical or chemical studies. 

The initial products of proteolytic digestion are free amino acids and small peptides. Further digestion of pep-... 

The probes of this type were shown to selectively label at least 75% of human kinases in crude cell lysates, thus demonstrating their selectivity and promiscuity for kinases [101]. As a follow up, the labeled kinases were subjected to proteolytic digestion, and the biotinylated peptides purified on avidin beads and analyzed by LC-MS/MS. This analysis demonstrated that the site of probe labeling was indeed the conserved active-site lysine as predicted. In contrast to the promiscuity demonstrated by the acyl phosphate probes, several selective covalent inhibitors of protein kinases have been used as ABPP probes. Wortmannin is a natural product derived from the fungus Penicillium funiculosum. It is a potent and specific covalent inhibitor of phosphoinositide 3-kinase (PI3K) and the PI3K-related kinase (PIKK) families [102, 103]. The use of natural products in relation to ABPP is covered by Breinbauer et al. [104]. 

The in vitro gastric and intestinal digests of selenized yeast food supplements were investigated by successive HPLC-ICP-MS and HPLC-ES-MS/MS [51]. The main compound extracted by both gastric and intestinal juice was Se-methionine, which was also the main Se compound extracted by proteolytic digestion from the yeast supplements. Two other minor compounds could be identibed as Se-cystine and Se(0)-methionine, a degradation product of Se-methionine [51]. 

Cell-free synthesis is often used to express troublesome proteins. For example, it is weU-suited to the expression of toxic proteins because there is no need to consider the action of a gene product on the viability of a host cell. The open nature of cell-free methods makes them useful for preparing proteins prone to mis-folding, aggregation into inclusion bodies, and proteolytic digestion. This is because the expression reaction conditions can be readily manipulated to promote proper protein folding and inhibit proteolysis. In addition. 

Production of pepsin and other proteolytic digestive enz)unes must be carefully controlled because the active enzymes would digest and destroy the cell that produces them. Thus, the stomach lining cells that make pepsin actually synthesize and secrete an inactive form called pepsinogen. Pepsinogen has an additional forty-two amino acids in its primary structure. These are removed in the stomach to produce active pepsin. 

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