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CPMV Versus TMV

1995, Nature Biotechnology

The story that Dr. Ricketts had worked at the germ warfare lab was a legend springing from the fact that he had bad breath.

• © 1995 Nature Publishing Group http://www.nature.com/naturebiotechnology /CORRESPONDENCE The story that Dr. Ricketts had worked at the germ warfare lab was a legend springing from the fact that he had bad breath. CPMV Versus TMV To the editor: The data presented by Turpen et al. in "Malarial Epitopes Expressed on the Surface of Recombinant Tobacco Mosaic Virus (TMV)" (Bio/Technology 13:53-57, January) were impressive. However, Harvey Bialy's claim (Bio/Technology 13:22-23, January) that Turpen et al. were the first to design chimeric viral coat proteins capable of reconstitution in infectious particles is incorrect. We have been developing cowpea mosaic virus (CPMV) as a presentation/delivery system for immunologic peptides (Virology 197:336-374, I 993; Virology 202:949-955, 1994) since 1992, and have demonstrated several advantages of CPMV over TMV for use in vaccines and therapeutic drugs. To date, we have engineered over 20 CPMV chimeras, with the largest insert being 42 amino acid residues (cf. 12 amino acids for TMV). CPMV chimeras give an unreduced yield of 1-2 g/kg fresh weight (cf. decreased yield with TMV chimeras). Furthermore, CPMV and its chimeras can be crystallized, facilitating structural studies. The IMAGE high-resolution UNAVAILABLE data collected from crystals of FOR COPYRIGHT a CPMV chimera REASONS containing an epitope from human rhinovirus14 will allow us to determine the structure of the insert, and to optimize the active conformation by exchanging specific amino acid residues. Structural studies of TMV are more difficult and time consuming. We also have strong data to show that CPMV chimeras containing HIV peptides produce a specific and desired immunological response in mice in that they raise higher levels of neutralizing antibodies (unpublished data, McLain et al.,AJDS Research and Human Retroviruses, in press). Lisa Ann Wisniewski* Nigel J. Dimmock George P. Lomonossoff John E. Johnson *Axis Genetics, Babraham Cambridge CB2 4AZ, U.K. Freeze/Thaw Isolation To the editor: Johnson and Hecht (Bio/Technology 12: 13561360, December) report an ingenious method for the selective isolation of recombinant proteins by re- 200 BIO/TECHNOLOGY VOL. 13 MARCH 1995 peatedfreeze/thaw cycles that apparently do not lyse the cell. Even allowing for the limitations of the method, e.g., proteins must be soluble and not exceed a certain molecular weight, and also allowing for the general tendency of researchers to "accentuate the positive" by publishing their best results, the freeze/thaw isolation process is clearly a major advance. What a pity then that the authors fall into the same trap as most other life scientists and pharmaceutical technologists: Many column inches are devoted to detailed descriptions of bacterial strains, expression vectors, suspension buffers, while the crux of the method, the freeze/thaw process, is disposed of in a few words. All we are told is that "pelleted cells were frozen by submerging .. .in a dry ice-ethanol bath for two minutes." A cookbook analogy would be a cake recipe that goes into great detail about ingredients and how they are mixed to create a dough, but disposes of the rest of the process by saying" ... and then it is baked in an oven." No information about the size of the baking pan, its proper location in the oven (top or bottom), no temperature, and no baking period. Eventually, with much trial and error, one not so skilled in the art will produce an acceptable cake and will have learned that its quality depends not only on the ingredients but on the process conditions. The same applies to freezing. Protein recovery could most like! y by maximized by proper control of freezing which is a very complex process, especially as applied to cells. It is governed by coupled heat and mass transfer, by solute and water fluxes (at different rates) across the plasma membrane, and by the osmotic conditions. Most researchers concentrate their attention on socalled ice formation, whereas the most dramatic effects are due to large increases in the concentrations of all soluble substances remaining in the residual liquid phase, which decreases in volume as freezing proceeds. Indeed, the freeze-concentration process can lead to the partial or complete inactivation of proteins and can be accompanied by chemical reactions, the mechanisms and rates of which differ from those normally studied in dilute solutions. Incidentally, immersing a frozen mixture in an ice/water bath does not ensure thawing at 0° C, as stated by the authors. Felix Franks Pafra Biopreservation 150 Cambridge Science Park Cambridge CB4 4GG, U.K. Biotech Obsolescence To the editor: The answer to the question raised in your editorial "Are Biotech Companies Obsolete?" (Bio/Technology 11:1047, November) is an emphatic no. The role of the biotech company in drug development is greater than ever-and growing every day. Entrepreneurial biotech companies spend a much larger percentage of their revenues on the R&D for