Expression of active thrombopoietin and identification of its key residues responsible for receptor binding.
In this report expression of the biologically active N-terminal half (amino acids 1-153) of thrombopoietin (TPO153) in Escherichia coli is described and the structure-function relationships in TPO are explored. TPO153 was chosen for expression because of its full biological activity. Since natural TPO153 cDNA expressed poorly, synthetic cDNA was constructed with a unique polymerase chain reaction to enhance the expression. In addition, the 5'-end codons of the synthetic cDNA were altered to maximize the expression. The expressed TPO153 was refolded and then purified to homogeneity. The protein is biologically active, and interestingly, the EC50 of this protein is 8-10-fold smaller in a TPO-dependent cell proliferation assay than that of full-length wild-type TPO. In order to identify the amino acid residues that are involved in the interaction between TPO and its receptor, all charged residues and some of the uncharged residues on the four putative helices of TPO were mutated and biological activities of the mutant proteins were examined. The mutagenesis studies suggest that there are at least two clusters of residues that are vital for TPO to be able to interact with its receptor. These residues are centred respectively around arginine 10 on helix 1 and around lysine 138 on helix IV. The successful expression of the protein in E. coli will greatly facilitate biochemical and crystallographic studies of TPO, and the structure-function relationship studies suggest that TPO has two binding sites which may interact with two individual receptors, resulting in dimerization of the receptors.
Hou
J
J
Arris Pharmaceutical Corporation, South San Francisco, CA 94080, USA. jinzhao_hou@arris.com
Zhan
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009363:Neoplasm Proteins; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D013926:Thrombopoietin / Q000235:genetics* / Q000378:metabolism*
D018076:DNA, Complementary; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; C082218:MPL protein, human; D013926:Thrombopoietin