Ge et al. 10.1073/pnas.0704179104. |
Fig. 6. E214A-BMP1 blocks cleavage of procollagen I by wild-type BMP1. Type I procollagen, metabolically radiolabeled with [3H]proline, was preincubated in the presence (+) or absence (-) of E214A BMP1 before incubation in the presence or absence of wild-type BMP1. Samples were subjected to SDS/PAGE and autofluorography.
Fig. 7. BMP1 cleavage sites within PRL and GH. Shown is an alignment of the sites at which BMP1 cleaves PRL and GH with sites at which BMP1 has previously been shown to cleave other substrates (1). Included in the alignment are murine (mur) and rat PRL and GH sequences corresponding to demonstrated cleavage sites in human (hum) PRL and GH and the predicted human PL cleavage site based on full-length sequence alignments of human PL with human GH and PRL. Aspartate residues conserved at the P1' positions of the various scissile bonds are in boldface, as are methionines and residues with aromatic side chains, noted as being N-terminal to scissile bonds in many previously identified BMP1 substrates.
1. Ge G, Greenspan DS (2006) Birth Defects Res C Embryo Today 78:47-68.
SI Materials and Methods
Protein Expression and Purification.
Flag-tagged BMP1, mTLD, mTLL1, and mTLL2 were expressed and purified as described (1). For E214A BMP1, two separate PCR amplifications were performed with primer sets 5'-GTTCCAGGAAACACTTCTAC-3' (forward) and 5'-TGGCCCAGCGCGTGGACCACAATGCCGA-3' (reverse), and 5'-TGGTCCACGCGCTGGGCCACGTCGT-3' (forward) and a T7 primer, yielding 388- and ~330-bp amplicons, respectively. The forward primer of the second amplification, carrying the mutation, has a 19-base overlap with the reverse primer of the first amplification. The two overlapping amplicons were combined and used as template to amplify an ~700-bp amplicon using the first forward and T7 primers. The band-purified 700-bp amplicon was cut with NsiI and AatII, and a resulting 432-bp fragment was used to replace corresponding wild-type sequences in BMP1 cDNA in pcDNA4/TO (Invitrogen). BMP1 sequences were fused to BM40 signal peptide sequences and had a C-terminal Flag epitope, as described for other recombinant BMP1 forms (1).
Primers 5'-GATCCATATGTTGCCCATCTGTCCCGGCG-3' (forward) and 5'-GATCAAGCTTAGCAGTTGTTGTTGTGGATGAT-3' (reverse) were used to PCR-amplify PRL cDNA from a human placenta cDNA library (Clontech). The product was cloned between NdeI and HindIII sites in pRSET B (Invitrogen). PRL truncated at Ala-159 (PRLdel159) was generated by site-directed mutagenesis in which the Asp-160 codon was converted to a stop codon. A point mutation for a Cys58Ala substitution was introduced to prevent incorrect disulfide bonding in PRLdel159. Expression was induced in Escherichia coli BL21(DE3) with isopropyl b-D-thiogalactopyranoside, and inclusion bodies were harvested, washed four times in PBS/0.5% Triton X-100 to remove endotoxins, washed once in PBS, and solubilized for 10 min in 8 M urea/20 mM ethanolamine-HCl (pH 10)/1% 2-mercaptoethanol/1 mM PMSF at 55°C and overnight at 4°C. After renaturation by extensive dialysis against 20 mM ethanolamine-HCl (pH 9) proteins were purified on a HiTrap Q column (Pharmacia), and endotoxin levels were determined to be <0.0002 unit/ng via the E-Toxate amoebocyte lysate assay (Sigma). Western blotting used 1:1,000 dilutions of primary antibodies and previously described conditions (2).
Chorioallantoic Membrane (CAM) Assay.
The CAM assay was conducted as reported (3). Briefly, 3-day-old chick embryos were removed from shells and incubated for 3 days in plastic Petrie dishes. On embryonic day 6, methylcellulose disks containing protein samples were applied to CAM surfaces above the dense subectodermal plexus. After 48 h eggs were examined under a dissecting scope (C60) and photographed.
1. Scott IC, Blitz IL, Pappano WN, Imamura Y, Clark TG, Steiglitz BM, Thomas CL, Maas SA, Takahara K, Cho KW, et al. (1999) Dev Biol 213:283-300.
2. Ge G, Seo NS, Liang X, Hopkins DR, Höök M, Greenspan DS (2004) J Biol Chem 279:41626-41633.
3. Fernández CA, Butterfield C, Jackson G, Moses MA (2003) J Biol Chem 278:40989-40995.