Schematic representation of MESA and 4.1R fragments and purified GST fusion proteins. (A) Schematic of the amino terminal region of MESA and the relative locations of the various MESA fragments used in this study. The first exon of the MESA gene encodes a putative signal peptide containing a hydrophobic core that is thought to be cleaved from the mature MESA polypeptide.6 The second exon encodes a highly charged protein that is characterized by 7 peptide repeat regions,6 of which only the GESKET peptide repeat region is shown. Amino acid (aa) residue numbers are shown adjacent to the MESA fragment names in the protein schematic. Previously, MESA fragment 3 (MF3) and MF4 have been described as binding and nonbinding regions for 4.1R, respectively, with MF3 containing the sequence (DHLYSIRNYIECLRNAPYI) mapped as the 4.1R binding region.3,7 Oligonucleotide primer sequences used in the construction of the MESA DNA fragments in this study are as follows: MF3(S)+19 and MF3(S)Δ19 (F), cgc gga tcc GAT ATC TAT ACG AAT TGT; MF3(S)+19 and MF3(S)Δ19 (R), ccg gaa ttc CAT TAC ATT CAC ATG TTT TCT A; MF4(S) (F), cgc gga tcc GCT AAT ACT GAA AAA AAT GAT; and MF4(S) (R), ccg gaa ttc ACT TGT TTT TTA ATT TCT TC. A MF3Δ19 DNA fragment was originally amplified by splice-overlap extension PCR to delete the region encoding the 19-residue sequence7 and was later used as a template to amplify the shorter MF3(S)Δ19 fragment. Sequence shown in upper case is complementary to the sequence amplified; lower case sequence is noncomplementary. Forward and reverse primers are designated F and R, respectively. (B) Schematic of the full-length 4.1R molecule and the relative locations of the various 4.1R fragments used in this study. Amino acid (aa) numbers are shown adjacent to the 4.1R fragment names and the protein schematic. Oligonucleotide primer sequences used in the construction of the recombinant 4.1R DNA fragments in this study are as follows: 30-kDa F1 and F2 (F), GGG CTG GCA AGC CAC GTT TGG TG; 30-kDa F1 and F2 (R), ccg gaa ttc TGT AAA ATT CCA AGG GAC; 30-kDa F3 (F), cgc gga tcc TTT AAT GTA AAG TTT TAT CC; 30-kDa F3 (R), ccg gaa ttc CGG GGC CAG TTT AAA ATC; 30-kDa F4 (F), cgc gga tcc AAT CAG ACC AAG GAA CTT; 30-kDa F4 (R), ccg gaa ttc GCG GTT AAT TCT CAG CTT; 30-kDa F5, F6, and F7 (F), cgg gat ccA TGA CTC CAG CTC AGG CT; 30-kDa F5 and F6 (R), gag cgc tcg agt caA AAT TTG GAT CCT AGC GCA AGA AAT TTG CTT TTG GG; 30-kDa F7 (R), gct agc TCG AGT CAC AAG TCC TTT GCT TTA TGA AG; 30-kDa F8 (F), cgg gat ccC AAG AGC AGT ATG AAA GTA CC; 30-kDa F8 (R), gag cgc tcg agt caA AAA TTT GGA TCC TAG CGC AAG AAA TTT GCT TTT GGG; 30 + 16-kDa (F), ccg gaa ttc TAA TGC ACT GCA AGG TTT CT; 30 + 16-kDa (R), ccg ctc gag TGG CTC AGC TTG CTC AGG; 22/24-kDa (F), cgc gga tcc CCT CCC CTG GTG AAG ACA C; and 22/24-kDa (R), gcc caa gct tCT CAT CAG CAA TCT CGG T. Construction of the pGEX-KG plasmids encoding the 30-kDa, 30-kDa F1, 16-kDa, and 10-kDa regions of 4.1R has previously been described.8 (C) RBC 4.1R and recombinant GST fusion proteins were purified as previously described.3,9 Each purified protein (approximately 1-2 μg total protein) was resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in 15% (wt/vol) gels before staining with Coomassie brilliant blue. The protein samples are 80-kDa RBC 4.1R (lane 1), GST-30 + 16 kDa (lane 2), GST-30 kDa (lane 3), GST-16 kDa (lane 4), GST-10 kDa (lane 5), GST-22/24 kDa (lane 6), GST–30-kDa F6 (lane 7), GST–30-kDa F5 (lane 8), GST–30-kDa F7 (lane 9), GST–30-kDa F8 (lane 10), GST (lane 11), GST–30-kDa F2 (lane 12), GST–30-kDa F3 (lane 13), GST–30-kDa F4 (lane 14), GST-MF3(S)+19 (lane 15), GST-MF3(S)Δ19 (lane 16), and GST-MF4(S) (lane 17).
