Polypeptides purification

Figures 13.54 thru 13.56 have been singled for special mention because of the unusual nature of the applications presented. Because peptides are also polyamides and because we routinely run polyamides using hexafluoro-2-pro-panol (FIFIP) as solvent, a modified polypeptide, poly (leucine-comethyl glutamate), was run in FIFIP. Figure 13.54 shows the results and suggests that modified HFIP might well prove to be a good solvent for at least some polypeptide purifications/characterizations. Figure 13.55 demonstrates the use of modi-...

Smith, J.C., Derbyshire, R.B., Cook, E., Viney, J., Brewer, S.J., Sassenfeld, H.M. and Bell, L.D. (1984) Chemical synthesis and cloning of a poly-arg gene fragment designed to aid polypeptide purification. Gene, 32. 321-327. 

The problem of polypeptide purification is an enormous one, and attempts at its solution consume many days in the laboratory. Several techniques can separate polypeptides on the basis of size, solubility in a particular solvent, charge, or ability to bind to a support. Although detailed discussions are beyond the scope of this book, we shall briefly describe some of the more widely used methods. 

The major disadvantage of solid-phase peptide synthesis is the fact that ail the by-products attached to the resin can only be removed at the final stages of synthesis. Another problem is the relatively low local concentration of peptide which can be obtained on the polymer, and this limits the turnover of all other educts. Preparation of large quantities (> 1 g) is therefore difficult. Thirdly, the racemization-safe methods for acid activation, e.g. with azides, are too mild (= slow) for solid-phase synthesis. For these reasons the convenient Menifield procedures are quite generally used for syntheses of small peptides, whereas for larger polypeptides many research groups adhere to classic solution methods and purification after each condensation step (F.M. Finn, 1976). 

Until the early 1960s, laboratory iavestigators rehed on dialysis for the separation, concentration, and purification of a wide variety of biologic fluids. Examples iaclude removal of a buffer from a proteia solution or concentrating a polypeptide with hyperosmotic dialysate. Speciali2ed fixtures were sometimes employed alternatively, dialysis tubes, ie, cylinders of membrane about the si2e of a test tube and sealed at both ends, were simply suspended ia a dialysate bath. In recent years, dialysis as a laboratory operation has been replaced largely by ultrafiltration and diafiltration. 

Polypeptides. These are a string of a-amino acids usually with the natural 5(L) [L-cysteine is an exception and has the R absolute configuration] or sometimes "unnatural" 7f(D) configuration at the a-carbon atom. They generally have less than -100 amino acid residues. They can be naturally occurring or, because of their small size, can be synthesised chemically from the desired amino acids. Their properties can be very similar to those of small proteins. Many are commercially available, can be custom made commercially or locally with a peptide synthesiser. They are purified by HPLC and can be used without further purification. Their purity can be checked as described under proteins. 

Classical gel electrophoresis has been used extensively for protein and nucleic acid purification and characterization [9, 10], but has not been used routinely for small molecule separations, other than for polypeptides. A comparison between TLC and electrophoresis reveals that while detection is usually accomplished off-line in both electrophoretic and TLC methods, the analyte remains localized in the TLC bed and the mobile phase is immediately removed subsequent to chromatographic development. In contrast, in gel electrophoresis, the gel matrix serves primarily as an anti-... 

Lyons, R.E., Lesieur, E., Kim, M., Wong, D., Brownlee, A., Pearson, R., and Elvin, C., The development of synthetic genes encoding repetitive resilin-like polypeptides Construct design, bacterial expression and rapid purification. Protein Eng. Design Select., 20(1), 25-32, 2007. 

There are still many challenging issues that remain to be addressed in the field of synthetic polypeptides. NCA purification has been one of the bottlenecks limiting the availability and scale-up of NCA monomers. Recrystallization has long been the... 

Wu WY, Fong BA, Gilles AG, Wood DW (2009) Recombinant protein purification by selfcleaving elastin-like polypeptide fusion tag. Curr Protoc Prot Sci 26.4 21-18... 

Chemical techniques for the isolation, purification and elucidation of the structure of toxins have evolved to the extent that it is frequently a routine procedure to identify the chemical nature of a newly discovered toxin once it has been purified, although difficulties arise when the toxin is a very large polypeptide, protein, or a very complex organic molecule. However, it is sometimes found that a toxin becomes progressively more labile and stabilizing contaminants are removed by the purification processes. An example of this is Cyanea toxic material which becomes increasingly labile with each purification step 111). 

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