We demonstrated earlier the presence of an erythroid cell stimulating factor (ESF) in mouse serum that enhanced the erythropoietin-dependent erythroid cell proliferation in vitro. Employing a highly specific monoclonal antibody generated in our laboratory, we showed further that ESF had an essential role in normal erythropoiesis in vivo [

Blood 98, 296a, 2001
,
Exp Hematol. 31, Supp. 1, 165, 2003
]. In order to clone and express this protein, ESF was purified from mouse serum and on Western blotting using the monoclonal anti-ESF antibody the purified protein was stained as bands of ~62/64 kDa. These bands were analyzed by Mass Spectrometry. The data were processed using Protein Lynx 2.1 (Waters) and the resulting peak list was submitted for database searching on Mascot. One of the peptide sequences had a highest similarity with Quiescin Q6 (Q6). This protein was then BLAST searched for mouse DNA sequences in the NCBI database for nucleotides corresponding to the mouse Q6. We designed the primers to reclone Q6 ORF (Open reading frame) having restriction sites NCO1 and ECOR1. Amplified product was sequenced to confirm DNA fragment. Fragment was cloned in pGEMT vector and subsequently re-cloned in expression vector pET30a(+). After each cloning step, the orientation of the insert was analyzed using restriction enzyme digestion. His-tagged Q6 protein was over-expressed in E.coli BL21 Codon Plus (DE3)-RP cells. Western blot analysis using anti-ESF antibody identified 62/64 kDa-bands of Q6 in the soluble fraction of the bacterial extract. Further characteristics of this recombinant protein and its CFU-E enhancing properties are being investigated and will be reported. Thus we have identified ESF to be a unique protein, quiescin Q6 and successfully cloned and expressed it. Expressed protein is similar to ESF as regards its antigenic property.

Topics:
antibodies, cloning, dna, dna fragments, dna restriction enzymes, erythroid cells, erythropoiesis, erythropoietin, expression vector, mass spectrometry

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2005, The American Society of Hematology
2005
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