Polypeptide Expression Systems

Two types of expression systems based on plant RNA viruses have been developed for production of immunogenic peptides and proteins in plants epitope presentation systems (short antigenic peptides fused to the CP that are displayed on the surface of assembled viral particles) and polypeptide expression systems (these systems express the whole unfused recombinant protein that accumulates within the plant). 

Polypeptide Expression Systems 4.2.2.1 Tobacco Mosaic Virus... 

Most biochemical and biocatalytic studies have been performed with type I B VMOs. This is partly because of the fact that they represent relatively uncomplicated monooxygenase systems. These monooxygenases are typically soluble and composed of only one polypeptide chain. Expression systems have been developed for a number of type I BVMOs while no recombinant expression has been reported for a type II BVMO. Cyclohexanone monooxygenase (CHMO) from an Acinetobacter sp. NCIMB9871 was the only recombinant available BVMO... 

FIGURE 4.2 Schematic diagram of RNA virus expression vectors, (a) TMV as an epitope presentation system, and (b) a polypeptide presentation system. Dark diamonds represent foreign antigen/peptide. 

The assembly of polypeptide chains into functional fibrinogen molecules has been studied in several expression systems. The order in which the polypeptide chains are joined together by disulfide bonds has been determined and specific structural features that are important for assembly, such as the coiled-coil, have been identified (Xu et al., 1996). Substitution of the cysteines with serine showed that the interchain disulfide ring at the proximal end of the coiled-coil, in addition to the disulfides between the two halves of the molecule, are necessary for assembly of the two half molecules (Zhang and Redman, 1996). The distal interchain disulfide ring is not necessary for assembly of the two half molecules but is necessary for secretion. Disruption of intrachain disulfide bonds and deletions of portions of the polypeptide chains have revealed which... 

Lanford RE, Luckow V, Kennedy RC, Dreesman GR, Notvall L, Summers MD (1989), Expression and characterization of hepatitis B virus surface antigen polypeptides in insect cells with a baculovirus expression system, J. Virol. 63 1549-1557. 

It is currently unclear whether, based on the current state of the art, a patent following the above-mentioned example could be extended to recombinant derivatives of the native protein. One might argue that, once the native protein is known and accessible, it needs no inventiveness to sequence the amino acids for parts of this protein, synthesize the corresponding DNAs, use these as probes to identify and isolate the entire coding sequence of the protein, which is then inserted into a suitable expression system to produce the protein in any desired form and quantity. Experience, however, teaches that it still requires some non-obvious steps and usually more than a limited degree of experimentation (often even a stroke of luck) to get there and to achieve the desired utility with recombinant polypeptides. For a vaccine it may be necessary to find and express the important epitopes in an appropriate (still unknown) way and to develop adequate purification and further processing protocols (with unpredictable technical... 

The cDNA and corresponding primary amino acid sequences of several CPRs including rat , rabbit- , and human were obtained by the mid-1980s, and the development of Escherichia coli expression systems paved the way for detailed molecular characterization of the polypeptide through site-directed mutagenesis. The three-dimensional structure of rat CPR was determined by X-ray crystallography in 1997 by Kim and coworkers -, providing the structural prototype for dual flavin oxidoreductases. 

C-terminal peptide a-thioester, mUdly activated peptide ester acting as a valuable key intermediate for the synthesis/semi-synthesis of polypeptides and proteins by both chemical ligation and the Aimoto thioester approach. The synthesis of the peptide a-thioester (—r thioester) can be performed by standard SPPS using Boc- or Fmoc-based chemistry or, for larger target polypeptides, by application of intein-based bacterial expression systems. Peptide a-thioester synthesis can also be carried out based on an N-S acyl shift reaction mediated by a thiol ligation auxiliary [F. B. Perler, E. Adams, Curr. Opin. Biotechnol. 2000, 377 D. Swinnen, D. Hilvert, Org. Lett. 2000, 2, 2439 R. Quaderer, D. Hilvert, Org. Lett. 2001, 3, 3181 T. W. Muir, Annu. Rev. Biochem. 2003, 72, 249 T. Kawakami et al.. Tetrahedron Lett. 2005, 46, 8805 J. A. Camarero, A. R. Mitchell, Prot. Pept. Lett. 2005, 12, 723]. 

Staphylococcal nuclease, a DNA-hydrolyzing enzyme, is a single polypeptide chain of 149 amino acid residues it has no disulfide bridge and contains one Ca ion. This protein was originally isolated from Staphylococcus aureus, but the gene has been cloned and inserted into several expression systems. Various mutants of this enzyme have been used to generate biochemical and biophysical data on the structural and functional consequences of altering the protein amino acid sequence. 

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