Recombinant chemokines expression

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. 

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,... 

The use of recombinant baculoviruses provides a rapid and efficient method to express and purify recombinant proteins, including chemokines. Expression in baculovirus-infected insect cells has been successfully applied to produce recombinant active human CC and CXC chemokines from diverse organisms such as Xenopus, mouse, and man (Braun et al., 2002 ... 

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.

Usually, we do not determine the titer of recombinant virus, since this protocol works well for over 10 chemokines. Successful infection is easily confirmed by the morphology of the High Five cells. Uninfected cells are firmly attached and infected cells become round up. If you do not detect the expression of recombinant protein, you should determine the titer of the recombinant virus, and should optimize MOI and timing of harvest. 

In general, the production of good MAbs depends to a large part upon the quality, purity and availability of antigen. In the production of the anti-RANTES chemokine monoclonal antibodies described here, a pET-3 E. coli expression system was used that allowed the manufacture of large quantities of recombinant human RANTES protein (see Notes 1,2, and 3). The recombinant protein was also used as a positive control in Western and immunoprecipitation experiments performed at later stages of the characterization of the MAbs (1). 

This 35-kDa protein was confirmed as the vCKBP by pierforming the same assays with two recombinant Ws, W RPVA35 and W Lister A35 from which the fully functional gene had been inactivated by insertion of the LacZ gene. Neither of these viruses produced a secreted protein that bound to I-chemokine. Furthermore, when the W Lister 35-kDa ORF was expressed with a G-terminal 6xHis-tag in the baculovirus system, or as an Fc fusion protein from stably transfected mammalian cells, binding of the recombinant products to... 

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