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. 2019 Feb 5;294(13):5214–5227. doi: 10.1074/jbc.RA118.005828

Figure 1.

Figure 1.

Expression of full-length and truncated CrmD recombinant proteins. A, Clustal Omega alignment of the amino acid sequences of the vTNFRs CPXV CrmE (gene K3R, strain elephantpox, UniProt/TrEMBL accession number Q9DJL2), CPXV CrmC (gene CPXV191, strain Brighton Red, UniProt/TrEMBL accession number Q9YP87), ECTV CrmD (gene E6, strain Hampstead, UniProt/TrEMBL accession number O57300), and VARV CrmB (gene G2R, strain Bangladesh 1975, UniProt/TrEMBL accession number P34015) and the SCPs CPXV SCP-1 (gene CPXV218, strain Brighton Red, UniProt/TrEMBL accession number Q8QMN0), CPXV SCP-2 (gene CPXV014, strain Brighton Red, UniProt/TrEMBL accession number Q8QN47), CPXV SCP-3 (gene CPXV201, strain Brighton Red, UniProt/TrEMBL accession number Q8QMP4), ECTV SCP-2 (gene EVN012, strain Naval, UniProt/TrEMBL accession number A0A075IJA5), and ECTV SCP-3 (gene EVN184, strain Naval, UniProt/TrEMBL accession number A0A075ILK3). The signal peptides were removed before the alignment. Numbers at the end of each line indicate the amino acid position relative to the complete protein sequence. The sequences were split into two columns corresponding to the CrmD TNF-binding and SECRET domains as labeled above each column. The sequences of each CRD in the TNF-binding domain of vTNFRs are indicated with differently colored lines. Conserved cysteines residues are shown in black background. An FN motif conserved in the SCPs and in the CRD4 of VARV CrmB and ECTV CrmD is highlighted in red. B, schematic representation of the CrmD truncated proteins expressed by recombinant baculoviruses. The CrmD protein molecule is depicted in blue. The two domains of CrmD, “TNF-binding domain” and “SECRET,” are labeled above. Ellipses numbered from 1 to 4 represent the different CRDs in the TNF-binding domain. Below the blue diagram, the sequences included in the different CrmD truncated proteins (SECRET163, SECRET181, and CRD-CrmD) as well as in the full-length CrmD are represented as solid black arrows. Dashed lines represent the portion of CrmD excluded in each truncated protein. Amino acid positions pertinent for these four recombinant proteins are numbered above the CrmD blue diagram. C, Coomassie Blue–stained gels showing the analysis of 1 μl of the final concentrated protein stocks (indicated above each gel line) by SDS-PAGE. Molecular mass is indicated in kDa.