Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The production of recombinant proteins such as the fibroblast growth factors (FGFs) is the key to establishing their function in cell communication. The production of recombinant FGFs in E. coli is limited, however, due to expression and solubility problems. HaloTag has been used as a fusion protein to introduce a genetically-encoded means for chemical conjugation of probes. We have expressed 11 FGF proteins with an N-terminal HaloTag, followed by a tobacco etch virus (TEV) protease cleavage site to allow release of the FGF protein. These were purified by heparin-affinity chromatography, and in some instances by further ion-exchange chromatography. It was found that HaloTag did not adversely affect the expression of FGF1 and FGF10, both of which expressed well as soluble proteins. The N-terminal HaloTag fusion was found to enhance the expression and yield of FGF2, FGF3 and FGF7. Moreover, whereas FGF6, FGF8, FGF16, FGF17, FGF20 and FGF22 were only expressed as insoluble proteins, their N-terminal HaloTag fusion counterparts (Halo-FGFs) were soluble, and could be successfully purified. However, cleavage of Halo-FGF6, -FGF8 and -FGF22 with TEV resulted in aggregation of the FGF protein. Measurement of phosphorylation of p42/44 mitogen-activated protein kinase and of cell growth demonstrated that the HaloTag fusion proteins were biologically active. Thus, HaloTag provides a means to enhance the expression of soluble recombinant proteins, in addition to providing a chemical genetics route for covalent tagging of proteins.
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PMID:HaloTag is an effective expression and solubilisation fusion partner for a range of fibroblast growth factors. 2613 34

Fibroblast growth factor 18 (FGF18) is a member of the fibroblast growth factor family and important in cartilage growth and development. However, the mechanism by which FGF18 mediates its biological functions is still unclear. In our study, we expressed the rhFGF18 protein fused to a HaloTag, (Halo-rhFGF18). MTT assay results indicated that both rhFGF18 and Halo-rhFGF18 have similar biological activities in NIH3T3 cells. However, basic FGF and acidic FGF were more potent than both rhFGF18 and Halo-rhFGF18. Confocal imaging data indicated that the red fluorescence labeled Halo-rhFGF18 strongly bound to ATDC5 cells and stimulated their proliferation and differentiation, which suggests that glycosaminoglycans may be involved in mediating the biological effects of rhFGF18 in ATDC5 cells. Moreover, western blot results demonstrated that, in ATDC5 cells, ERK1/2 signaling is activated upon stimulation with rhFGF18. Our results may open doors for the use of rhFGF18 as a drug to promote cartilage growth.
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PMID:Expression of Halo-hFGF18 and study of its effect on differentiation of ATDC5 cells. 3041 1