Recombinant chemokines

Chemokines share another property which has been of help in their purification especially from natural sources (see Chapter 1 and [1-4] in that they all bind strongly to glycosoaminoglycans [GAGs]). Thus, they will bind to Heparin columns, and this property has been exploited in the purification of recombinant chemokines as well as the natural proteins. Since the chemokines are mostly highly basic with pi values around 9, this interaction with GAGs could be attributed to electrostatic interactions. However, MIP-la, which has an acidic pi of 4.7, yet still binds to Heparin columns, albeit less strongly than... 

After renaturation, the majority of the recombinant chemokines are easily quantified by UV spectroscopy, although there are examples of chemokines such as NAP-2, which do not possess an aromatic amino acid that serve as chromophores. In this case, quantification is achieved by comparison of the peak height on reverse phase HPLC analysis to that of a known concentration of another chemokine. They can then be lyophilized after a change of buffer into a trifluoroacetic acid or acetic acid solution, which facilitates their storage as lyophilized powders. It is important that they are redissolved in water, before dilution into buffer or medium. Their handling is easy and rapid, as they are instantly soluble at concentrations as high as 1 mM if necessary, in aqueous solutions. 

The amounts of total protein in crude extracts are estimated using a colometric assay such as the Coomassie Plus Protein assay reagent (Pierce) or equivalent. The amount of purified recombinant chemokine protein is determined by UV spectroscopy at 280 nm. The extinction co-efficient is calculated from the amino acid composition according to the formula ... 

Bell, M. D., Taub, D. D., and Perry, V. H., Overriding the brain s intrinsic resistance to leucocyte recruitment with intraparenchymal injections of recombinant chemokines, Neuroscience, 74, 283, 1996. 

Veazey, R. S., Ling, B., Green, L. C., Ribka, E. P., Lifeon, J. D., Piatak, M., Jr., et al. (2009). Topically applied recombinant chemokine analogues fuUy protect macaques from vaginal simian-human immunodeficiency virus chaUenge. The Journal of Infectious Diseases, 199, 1525-1527. 

Proudfoot, A. E., Borlat, F. (2000). Purification of recombinant chemokines fromE. coH. Methods in Molecular Biology, 138, 75-87. 

Already from the beginning of their discovery, recombinant chemokines were expressed in bacteria and shown to possess equivalent activity as their naturally expressed counterparts (Bindley et al., 1988). Several general protocols for purification are available (Edgerton, Gerlach, Boesen, Abet,... 

Production of Recombinant Chemokines Tagged with Fluorescent Proteins... 

Figure 1 Recombinant chemokine production reproduces the natively folded bioactive protein secreted from eukaryotic cells. (A) Folding, transport, and secretion of chemokines in eukaryotic cells. (B) Schematic diagram of expression, purification, and refolding of recombinant chemokines from coli. (C) The 3D structure of human CXCL12 illustrates the conserved chemokine fold with structurally important disulfide bonds and functionally important native N-terminus.

There are many approaches to producing functional recombinant chemokines from E. coli in addition to the ones referenced or presented here. The goal of this work is to describe a robust protocol we employ for many different chemokines, which draws from a variety of previously described approaches. We illustrate the impact of disulfide shuffling cocktails on the... 

For most chemokines, which contain four cysteines that form two conserved disulfide bonds, a measured mass to charge ratio will be four mass units less than the mass predicted based on protein sequence. Hence, measurement of a recombinant chemokine s mass to charge ratio using a sensitive and accurate mass spectrometer can confirm not only protein identity but also disulfide oxidation as seen in Fig. 4. 

The recurrent success of chemokines or chemokine variants in several animal models of human disease supports the potential of chemokines or chemokine-derived molecules for clinical use in medicine. Continued access to high quality recombinant chemokines that are produced for medicinal use is paramount to the advancement of their therapeutic uses. Additionally, clinical administration of chemokines would require that they be manufactured and formulated in facilities and according to protocols that satisfy the Current Good Manufacturing Practices set by the U.S. Food and Drug Administration. 

In conclusion, the supply of pure, natively folded chemokine proteins is vital for a variety of basic, translation and cHnical research programs. Our structure-function studies and drug development efforts have motivated the development of the robust protocol for recombinant chemokine expression, refolding, purification, and vahdation presented here. Finally, many additional factors must be considered when manufacturing a product under cGMP guidehnes that may not be obvious to personnel in research settings. 

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