The UCMD-Causing COL6A1 () Intron Mutation Leads to the Secretion and Aggregation of Single Mutated Collagen VI α1 Chains

Figure 4

Purification and characterization of recombinantly expressed full-length α1 wild type and mutant chains. (a) Immunoblot with Ponceau S staining of supernatants from not transfected (nt) HEK293 EBNA cells, cells transfected with the wild-type chain (WT) or the mutant chain (Pex) detected with an anti-Strep antibody. (b) Coomassie-stained SDS-PAGE under nonreducing (-βMe) and reducing conditions (+βMe) after affinity purification. (c) Size exclusion chromatography of affinity purified samples. (d) Secondary structure analysis by circular dichroism spectroscopy and (e) Coomassie-stained SDS-PAGE gel of purified proteins and negative staining electron microscopy of single particles. Particles marked by white frames are further magnified. Scale in overviews and 10 nm in magnified images. The results indicate that the mutant chains are expressed and secreted and do not show gross structural aberrations. Under these conditions, both wild type and mutant chains tend to aggregate, but the aggregates formed by the mutant chains are larger.

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