Techniques, disulfide bond formation

Once the structural features of a reference standard of the desired protein have been well characterized, lot-to-lot confirmation of identity can be conducted using a carefully selected group of tests, wherein the lot undergoing analysis is compared to the reference standard. Tests commonly employed for this purpose are listed in Table III. Peptide mapping is perhaps the most powerful and universally used technique since it provides relatively specific confirmation of correct primary sequence and, when non-reducing conditions are employed, can be used to confirm correct disulfide bond formation. Tertiary structure is difficult to address directly on a routine (lot-to-lot) basis, and the presence of correct biological activity is often used as evidence that the correct tertiary structure is maintained. 

Cyclization is one of the earliest techniques applied to design peptidomimetics. Cyclic peptides are more stable to amide bond hydrolysis and allow less conformational flexibility consequently, the resulting analogs are anticipated to be more selective and less toxic. Methods for restricting conformations include peptide backbone cyclization, disulfide bond formation, side-chain cyclization, and metal ion chelation. 

After peptide chain extension and disulfide bond formation by oxidative cyclization, the phenylacetyl group could be removed enzymatically in 74% yield. The technique has been applied further in the synthesis of even larger peptides, for example in the deprotection of tris(phenylacetamido)porcine insulin.P l Since PGA is commercially available and devoid of any peptidase activity, this method appears to be generally useful for the construction of lysine-containing oligopeptides. 

Mass spectrometry, in addition to RP-HPLC, serves as a powerful technique for assessing disulfide bond formation. Additionally, tandem mass spectrometry (MS-MS) can in some instances be used to define the pairing of cysteines, like the non-conserved disulfide bond in CCL28 that finks C30 to C80 (Thomas et al., 2015), or confirm the locations of posttranslational modifications, like pyroglutamate formation in CCL2 as shown in Figs. 4 and 5, respectively. 

Various cyclization techniques have been used in the past, which include disulfide bond [61], side-chain lactam [59], and head-to-tail amide bond formation [62], In most cases, the readiness of an open-chain precursor to cyclize depends on the size of the ring and the sequence of the peptide to be cyclized. Usually, ring closure with hexa- and pentapeptides is somewhat hampered however, the cyclization may be enhanced by the presence of turn structure inducing amino acids such as glycine, proline, and D-amino acids, etc., as was the case in the study of McBride et al. [63], Spatola et al. [64] have recently conducted extensive studies in these aspects and several cyclic penta-, hexa-, and heptapeptide libraries were prepared and analyzed. Their results showed that rapid cyclization rates can be achieved with optimized synthesis and cyclization procedures, and many combinations of cyclic peptides can be formed in high quality if they contain structural features that make cyclization more facile. 

Proteins that contain multiple cysteines are difficult to refold after high-level production in E. coli because of the formation of incorrect disulfide bonds. Replacement of individual cysteines or specific disulfide bonding pairs without compromising the functional activity of the protein can result in increased yield of the correctly folded protein. This technique has been applied successfully to interleukin-2 (IL-2 Wang et al, 1984), human fibroblast interferon (Mark et al, 1984), and basic fibroblast growth factor (b-FGF Rinas et al, 1992). 

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