Protein recombination synthetic method

Flavor notes and products of lipid oxidation in cooked meats, 85,86/ Flavor peptides convenient synthesis, 149-156 enzymatic synthesis, 151,154-156 protein recombination synthetic method, 149... 

There is a tendency to reserve semisynthetic and totally synthetic methods for the introduction of bonds and residues that cannot be specified by the genetic code. The present chapter will concentrate on these aspects. However, semisynthesis can have a role to play even when building structures that are completely accessible to the genetic code. The first industrial challenge for the emerging technologies of total chemical synthesis, recombinant protein expression, and semisynthesis was the economic production of human insulin in pharmaceutically usable quantity and quality. The semisynthetic human insulin that was made from porcine insulin proved exceptionally convenient to produce, and was the first introduction to human insulin for very many patients. 

Today, it is well-known that peptides or proteins exhibit various kinds of taste. Our group has been researching on the relationship between taste and structure of peptides, BPIa (Bitter peptide la, Arg-Gly-Pro-Pro-Phe-Ile-Val) (7 as a bitter peptide, Om-p-Ala-HCl (OBA), Om-Tau-HCl as salty peptides(2j, and "Inverted-Aspartame-Type Sweetener" (Ac-Phe-Lys-OH) as a sweet peptide(5). The relationship between taste and chemical structure was partly made clear. Since commercial demand for these flavor peptides is increasing, we need to develop new synthetic methods which can prepare these peptides in large scale. We developed the following two methods (1) protein recombination method as a chemical method, (2) enzymatic synthesis using chemically modified enzyme as a biochemical method. 

Recombinant DNA drug production has been a success story, but the technology does have its drawbacks and disadvantages. For example, the use of bacterial plasmids and, less commonly, viruses, as vectors for the insertion of genes into host cells has its limitations. While such methods work well for smaller drug molecules, such as insulin, they are much less effective in the production of larger, more complex proteins that constitute many of the drugs that chemists would like to manufacture synthetically. 

This operation is illustrated by work on the human cytokine GCSF, a protein of 174 residues. The disulfide-linked loop (64-74) of this protein, thought to be involved in binding to the receptor, was replaced by various non-coded synthetic structures.[10] In this case recombinant methods were used to produce a variant of the parent protein having the appropriate cleavage sites for the production of the wanted fragments. 

The use of CE methods for routine quality control of synthetic or recombinant peptides-proteins necessitates optimization strategies for rapid method development. Ideally, the methods should be simple, fast, and robust. Because capillary electrophoresis in the zone format is the most simplistic, initial efforts should be directed toward the use of a simple buffer system [61]. The high efficiency and reproducibility in protein-pep-tide separations demands that interactions between the analyte and capillary wall be neglible. The use of low-pH buffers generally results in enhanced reproduciblity, and hence ruggedness, as slight variations in the capillary surface will have little impact on the already suppressed EOF. 

Such approaches underpin the current popularity of RP-HPLC procedures for the purification of synthetic or recombinant polypeptides at the production scale, or analogous approaches employed in the HP-IEX of commercially valuable proteins. However, in some cases when linear scale-up methods are applied to higher molecular weight polypeptides or proteins, their biological activity may be lost due to unfavorable column residency effects and sorbent surface area dependencies. It is thus mandatory that the design and selection of preparative separation system specifically address the issues of recovery of bioactivity. Often some key parameters can be easily controlled, i.e., by operating the preparative separation at lower temperatures such a 4°C, or by minimizing column residency times. 

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