Mapping of a candidate region for susceptibility to inclusion body myositis in the human major histocompatibility complex

 Inclusion body myositis (IBM) is a form of idiopathic inflammatory myopathy of unknown aetiology. A strong association with HLA class II (HLA-DR3) suggested a role for genes in the human major histocompatibility complex (MHC) in the predisposition to this disease. In this study, we have taken advantage of the ancestral haplotype (AH) concept and historical recombinations to map for a possible susceptibility gene(s) in the MHC. We performed detailed typing of three MHC-related HSP70 genes and defined allelic combinations in the context of MHC AH. We also modified existing methods to give a simple and accurate method for typing two TNF microsatellites. Using the HSP70 and TNF markers and HLA-DR, –B, and C4 typing of our patients with IBM, we defined a potential site for the MHC-associated susceptibility gene(s) in the region between HLA-DR and C4. Received: 16 July 1998 / Revised: 14 January 1999     

Inclusion body formation by interleukin-1β depends on the thermal sensitivity of a folding intermediate

We show that sequence and growth temperature effects on IB formation in the small, monomeric β-barrel protein interleukin-1β (IL-1β) can be quantitatively reproduced in an in vitro system in which IL-1β is refolded from denaturant at different temperatures. The results suggest that temperature and mutational effects on IB formation may be based on intrinsic properties of the protein sequence rather than interactions with chaperones or other cellular factors. We also report striking correlations of IB formation with mutation-dependent changes in residue hydrophobicity. The nature of these trends differs considerably with residue position, however, suggesting that they are mediated by particular local environments created by an ordered structure.     

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

Heterodimerizing peptides, such as the de novo designed E5/K5 peptide pair, have several applications including as tags for protein purification or immobilization. Recently, we demonstrated that E5-tagged epidermal growth factor (EGF), when bound to a K4 expressing adenovirus, promotes retargeting of the adenovirus to EGFR expressing target cells. In this study, we present the Escherichia coli expression, refolding and purification of human EGF fused with the E5-coil (E5-coil-EGF) or with the K5-coil (K5-coil-EGF). EGF receptor phosphorylation and cell proliferation assays demonstrated that the biological activity of the coil-tagged EGF versions was comparable to that of non-tagged EGF. Additionally, analysis of the binding of E5/K5-coil-EGF to cell surface EGFR or to soluble EGFR ectodomain, as measured by cell-based binding competition assays and by SPR-based biosensor experiments, indicated that the coil-tagged EGF versions bound to EGFR with affinities similar to that of non-tagged EGF. Finally, we show that E-coil-tagged EGF, but not non-tagged EGF, can retarget a K-coil containing adenovirus to EGF receptor expressing glioblastoma tumor cells. Overall these results indicate that E. coli expression offers a practical platform for the reproducible production of fully biologically active E5/K5-coil-tagged EGF, and support applications of heterodimerizing coil-tagged ligands, e.g. the targeting of viruses or other entities such as nanoparticles to tumor cells, or growth factor immobilization on cell culture scaffolds for tissue engineering.