Recombinant chemokines purification

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... 

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,... 

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.

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. 

For production of recombinant fluorescent chemokine fusion proteins Trichoplusia ni 5B1-4 cells (H5 high five cells) are grown at 27 °C in suspension cultures in Insect-Xpress medium in 1800 mL Fembach culture flasks. Importandy, the cells grow without FCS facilitating purification of the chemokine from the culture supernatant. 

Purification of Recombinant Fiuorescent Chemokine Fusion Proteins... 

Purification of Recombinant Tagged Chemokines from Bacteria... 

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