A Necessary Evil?

John C Ford^*

Department of Chemistry, Indiana University of Pennsylvania, USA

*Corresponding Author:

John C Ford, Ph.D
Department of Chemistry
Indiana University of Pennsylvania
USA Indiana, PA 15705, USA
E-mail: jford@iup.edu

Received date: May 06, 2013; Accepted date: May 08, 2013; Published date: May 10, 2013

Citation: Ford JC (2013) A Necessary Evil? J Anal Bioanal Tech 4:e115. doi: 10.4172/2155-9872.1000e115

Copyright: © 2013 Ford JC. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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As I write, Northeastern University (Boston, MA, USA) is preparing “a Celebration of Dr. Barry Karger’s 50 Years of Service”. Professor Karger has made numerous contributions to science, not the least of which is co-authoring (with Lloyd Snyder and the late Csaba Horvath) the influential text, An Introduction to Separation Science. I consider myself fortunate to have been in his research group during the late seventies and early eighties, thank him for the training he provided, and wish him continued success.

As a graduate student, Professor Karger’s courses were, of course, required and I still remember him elegantly deriving an expression showing that smaller particle sizes result in faster separations, provided the system is not pressure-limited. (To chromatographers, UHPLC was obvious, merely awaiting appropriate technology). Then, following perhaps twenty-five minutes of chalkboard and lecturing, he turned and said, “Of course, you only do chromatography if you have to”.

That caused considerable consternation among us graduate students. Prof. Karger’s point was, of course, that chromatography takes time and an adequately selective analytical technique would sense only the desired analyte(s), eliminating the need for separation, reducing the required time. Were we learning something not really desirable? Would companies shun us and our undesirable skills? A similar reaction accompanied the early reports of FT-MS and tandem quadrupole MS: why would anyone do chromatography when you could detect individual compounds in complex mixtures?

While I echo Prof. Karger’s words each semester in my own courses, the memory was brought clearly to mind recently when I read, “Non-chromatographic methods for protein purification are attractive in light of the expense and time required for chromatography” [1]. This sentence started a brief description of a report detailing a genetic engineering approach to avoiding chromatography in the purification of expressed proteins [2]. That article commenced with, “Nonchromatographic purification techniques are of significant interest since chromatography is typically the most expensive step in protein purification”, Another indictment against chromatography. Their system adds a peptide which forms a precipitate in the presence of Ca²⁺, pulling the desired gene product out of solution. Centrifuge, wash, redissolve with an EDTA solution...voilà! Purified product.

Genetic engineering approaches to ease separation difficulties are not new. Introduced in 1988, the now-common “His tag” coupled with metal-affinity chromatography was the first [3] of which I am aware. This eventually gave rise to metal chelate affinity precipitation [4,5], a direct precursor to the report mentioned above. Living systems are extremely complex, and give rise to extremely complex separation problems. While powerful, chromatography adds to that complexity. And, as pointed out, chromatography takes time.

Although broadly applicable, chromatography has always had a special relationship to biochemistry. Most of us are aware that Mikhail Twsett, the inventor of chromatography, was a botanist [6]. Perhaps less well known is that Martin and Synge, who were awarded the Nobel Prize in 1952 “for their invention of partition chromatography” [7], were working at National Institute for Medical Research in London, focusing on analytical questions of biochemical interest. The first published gas chromatogram was of volatile fatty acids [8]. And while the half of the 1972 Nobel Prize awarded to Stein and Moore was “for their contribution to the understanding of the connection between chemical structure and catalytic activity of the active centre of the ribonuclease molecule” [9], their development of the amino acid analyzer (essentially a specialized liquid chromatography) was also a significant achievement. Most texts on bioanalytical chemistry devote one or more chapters to chromatography [10], as do texts on protein purification [11]. Perhaps it’s a love-hate relationship, but chromatographyand biochemistry have long had a special relationship.

No one involved with protein purification is so naive as to think single-step purification will lead to a truly homogeneous product. The gels in [2] show faint, but visible contamination in the purified samples. Additional, likely chromatographic, purification will be necessary to get a clean product. Chromatography isn’t in danger of obsolescence.

However, it IS time-consuming and has limited sample capacity - that is, limited by the available funds, in any case. And, excepting the size-exclusion mode, chromatography involves a phase change, which necessarily increases the possibility of denaturation and sample loss. (Precipitation shares that possibility, by the way). Particularly for preparative applications, there is a need for a gentle, highly selective, non-chromatographic purification process. Given the modern approach of purifying sufficient protein to determine its gene sequence, then cloning the gene into an expression vector and isolating larger quantities of the desired protein from an expression system, the genetic engineering approaches are quite a step in the right direction. I expect the future will see more such systems.

In thinking about it, adding a peptide to a gene product is, in a sense, analogous to making a derivative of a small-molecule analyte. The “genetically-added” tag derivatizes the desired product to increase its difference from the matrix components. Old dog, new trick, as it were.

References

1. Lo P, Blow N (2013) Playing tag with proteins. Biotechniques 54: 180.
2. Shur O, Dooley K, Blenner M, Baltimore M, Banta S (2013) A designed, phase-changing RTX-based peptide for efficient bioseparations. Biotechniques 54: 197-206.
3. Hochuli E, Bannwarth W, Dobeli H, Gentz R, Stuber D (1988) Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent. Nature Biotechnology 6: 1321-1325.
4. Balan S, Murphy J, Galaev I, Kumar A, Fox GE, et al. (2003) Metal Chelate affinity precipitation of RNA and purification of plasmid DNA. Biotechnol Lett 25: 1111-1116.
5. Kumar A, Wahlund PO, Kepka C, Galaev IY, Mattiasson B (2003) Purification of histidine-tagged single-chain Fv-antibody fragments by metal chelate affinity precipitation using thermoresponsive copolymers. Biotechnol Bioeng 84: 494-503.
6. Ettre LS (2003) M. S. Tswett and the Invention of Chromatography. LC/GC 21: 458-467.
7. Martin AJP, Synge RLM (1952) The Nobel Prize in Chemistry 1952. Nobelprize.org.
8. James AT, Martin AJ (1952) Gas-liquid partition chromatography: the separation and micro-estimation of volatile fatty acids from formic acid to dodecanoic acid. Biochem J 50: 679-690.
9. Anfinsen C, Moore S, Stein WH (1972) The Nobel Prize in Chemistry 1972. Nobelprize.org.
10. Mikkelsen SR, Corton E (2004) Chapter 14 Chromatography of Biomolecules. Analytical Biochemistry, John Wiley & Sons, Inc., Hoboken, NJ, 268-293.
11. Scopes RK (1993) Chapters 5-7: Separation by Adsorption, in Protein Purification: Principles and Practice. (3rdedn), Springer-Verlag, New York, Inc., New York, 102-236.