Protease cleavage site specificity

A slightly modified approach was applied in the identification of protein complexes in Schizosaccharomyces pombe [120]. The major modification involves the use of a tandem affinity purification [121]. To a protein of interest, an epitope is appended that contains two different affinity tags separated by a specific protease cleavage site. This enables isolation of the complex under milder conditions. 

The simplest modification is to place a destabilizing amino acid at the N-terminal end according to the N-end rule (Varshavsky, 1992). This can be done by expressing the protein as a fusion protein, e.g. with maltose binding protein behind a cleavage site specific for a rare-cutting protease, such as factor Xq. In this case the N-terminal amino acid can be any amino acid except proline. 

Figure 2 A general overexpresion vector. Most Escherichia coii vectors for recombinant protein production will have most of these features. The N- and C-terminal fusions and associated protease cleavage sites are optional. The multiple cloning site (MCS) may be replaced by DNA sequences for site-specific recombination in some vectors.

Fig. 1 Schematics of tandem affinity purification (TAP). The methodology of using TAP technique for protein complex purification is outlined in 6 steps. (1) The target protein (the bait ) is co-transcribed with the tandem-arranged affinity tags to either the N- or the C-terminus of the gene (shown is the N-terminal fusion). The two affinity tags (shaded boxes 1 and 2j are separated by a protease cleavage site (blank bo)(j, from where the far-end tag would be cut off and the near-end one would be exposed to the affinity columns for a second-round purification. (2) The bait and the tags are expressed as a chimeric fusion protein. The bait would form a complex with its in vivo partners (depicted as A, B, and Q. Nonspecific association may happen as well, as depicted by D, E, and F. (3) The first round purification via specific interaction between the affinity binder 1 and the far-end tag eliminates most nonspecific associated proteins. (4) In the second round purification, the near-end tag is cut off and the protein complex is further purified, with most nonspecific associates removed. (5 and 6) The bait protein and its associated proteins are eluted from the column and are further analyzed either by using antibodies, or by mass spectrometry...

The primary analytical applications of RPLC in the development of biopharmaceuticals are the determination of protein purity and protein identity. Purity is established by analysis of the intact protein, and RPLC is useful in detecting the presence of protein variants, degradation products, and contaminants. Protein identity is most often established by cleavage of the protein with a site-specific protease followed by resolution of the cleavage products by RPLC. This technique, termed peptide mapping, should yield a unique pattern of product peptides for a protein that is homogeneous with respect to primary sequence. 

In the process of viral assembly, HIV PR specifically cleaves nine cleavage sites on GAG and GAG-POL polypeptides [21]. Examination of the amino acid composition of the recognized substrate sites (Table 1) indicates their hydrophobic character and significant sequence variability. The loose specificity of HIV PR most likely reflects its functions in a world of reduced complexity within the confines of the budding virion. The length of the viral protein precursors (approximately 1500 amino acids) reduces the number of potential sequences the protease must discriminate from in selecting its nine cleavage sites. Therefore,... 

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