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 de scribed and the structure-function relationships in TPO are explored, TPO153 was chosen for expression because of its full biological activi ty. Since natural TPO153 cDNA expressed poorly, synthetic cDNA was con structed with a unique polymerase chain reaction to enhance the expres sion. In addition, the 5'-end codons of the synthetic cDNA were altere d to maximize the expression. The expressed TPO153 was refolded and th en purified to homogeneity. The protein is biologically active, and in terestingly, the EC50 of this protein is 8-10-fold smaller in a TPO-de pendent cell proliferation assay than that of full-length wild-type TP O. In order to identify the amino acid residues that are involved in t he interaction between TPO and its receptor, all charged residues and some of the uncharged residues on the four putative helices of TPO wer e mutated and biological activities of the mutant proteins were examin ed. The mutagenesis studies suggest that there are at least two cluste rs of residues that are vital for TPO to be able to interact with its receptor. These residues are centred respectively around arginine 10 o n helix I and around lysine 138 on helix IV. The successful expression of the protein in E. coli will greatly facilitate biochemical and cry stallographic 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. (C) 1998 Academic Press Limited.