Tagged chemokine

Part A Expression and Purification of Fluorescent Protein-Tagged Chemokines from Baculovirus-Infected Insect Cells... 

Part B Expression and Purification of Tagged Chemokines from Bacteria for Enzymatic Labeling... 

Cloning of Tagged Chemokines-Encoding DMAs for Expression in coli... 

Purification of Recombinant Tagged Chemokines from Bacteria... 

Fig 2. Expression of chemokines with and without N-terminal tags. Murine RANTES and human herpesvirus 8 vMIP-lB were expressed either as mature proteins (native) or with the addition of the residues MKKKWPR- to the N-terminus (K3) using the T7 based expression vector pET24d in either BL21(DE3) or BL21(DE3)pLysS cells. Each lane of this Coomassie stained SDS-polyacrylamide gel contains 0.04 OD600 units of the indicated strain following a 3-h induction. 

We routinely clone chemokines using polymerase chain reaction (PCR). The small size of chemokine genes, the frequent addition of N-terminal or C-terminal tags, and the need to localize precisely the initiator methionine codon in bacteria expression systems are all most easily dealt with by amplifying the genes. In this section, it is assumed that the reader has a grasp of basic cloning techniques (18) and is familiar with simple PCR amplification (21). 

Identification of Novel Chemokines From Expressed Sequence Tag Databases... 

Wells, T. N. C. and Peitsch, M. C. (1997) The chemokine information source identification and chracterization of novel chemokines using the WorldWideWeb and expressed sequence tag databases. J. Leuk. Biol. 61, 545-550. 

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... 

In the past six years, a large number of new chemokines and chemokine receptors have been discovered and characterized. This is mainly attributed to the rapid progress in the area of bioinformatics and expressed sequence tag (EST) databases. It is now clear that chemokine function is not just restricted to cell attraction. Chemokines are also important in mediating the development and functional aspects of leukocytes. Recent discoveries provide convincing and compelling evidences supporting the roles of these messengers and their receptors in the resolution of viral infections and virus survival. 

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