Sequence comparison and analysis of HAPO. Schematic presentation of the exon structure of PRG4 and expressed fragment of HAPO (A) and DNA and deduced amino acid sequence of expressed fragment of HAPO (B). The expressed cDNA fragment containing 879 bp (293 amino acids) is a truncated form of HAPO on the basis of the N-terminal amino acid and molecular weight of natural HAPO purified from the urine of patients with aplastic anemia. Recombinant human HAPO is expressed by pET-22b vector as secreted protein in periplasm. (C) Expression and purification of His-tagged terminal HAPO protein. (i) A 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie blue. Lane 1 indicates crude extract of E coli BL21 (DE3) from periplasm (control); lane 2, crude extract of recombinant E coli BL21 (DE3) (pET22b⁺/HAPO) from periplasm before induction; lane 3, crude extract of recombinant E coli BL21(DE3) (pET22b⁺/HAPO) from periplasm after induction; M, protein marker. (ii) A 12% SDS-PAGE stained with Coomassie blue. M indicates protein marker; lane 1, step 2-purified recombinant HAPO by metal-chelating chromatography; lane 2, step 3-purified recombinant HAPO by SP sepharose Fast Flow (FF) chromatography.