PURIFICATION AND CHARACTERIZATION OF RECOMBINANT HUMAN GRANULOCYTE-COLONY-STIMULATING FACTOR (RHG-CSF) DERIVATIVES - KW-2228 AND OTHER DERIVATIVES

Various derivatives of recombinant human granulocyte colony-stimulatin g factor (rhG-CSF) have been overproduced in Escherichia coli with the strong, inducible trp promoter. A derivative designated as KW-2228 in which the amino acids were replaced at five positions showed more pot ent granulopoietic activity and stability than those of wild-type both in vitro and in vivo. The purification involved a sequential renatura tion process and three-step chromatography. Refolding succeeded in ver y high yield using a urea system. The purity of KW-2228 was greater th an 99% as measured by SDS-PAGE and HPLC analysis. According to circula r dichroism and nuclear magnetic resonance spectroscopy, rhG-CSF and K W-2228 have very similar conformations. This suggests that the substit ution of five amino acids does not appreciably change the conformation of hG-CSF. KW-2228 ([Ala1, Thr3, Tyr4, Arg5, and Ser17]-hG-CSF) and d erivative A ([Ala1, Thr3, Tyr4, Arg5]-hG-CSF) are easily crystallized and they show similar in vitro activity. On the other hand, neither rh G-CSF nor derivative B ([Ser17]-hG-CSF) are crystallized under the sam e conditions. Thus, the four amino acid substitution (Ala1, Thr3, Tyr4 , Arg5) of the N-terminal sequence may facilitate crystallization. The change of Cys17 to Ser may not influence the stability and activity o f hG-CSF.