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-stimulating 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 potent granulopoietic activity and stability than those of wild-type both in vitro and in vivo. The purification involved a sequential renaturation process and three-step chromatography. Refolding succeeded in very high yield using a urea system. The purity of KW-2228 was greater than 99% as measured by SDS-PAGE and HPLC analysis. According to circular dichroism and nuclear magnetic resonance spectroscopy, rhG-CSF and KW-2228 have very similar conformations. This suggests that the substitution of five amino acids does not appreciably change the conformation of hG-CSF. KW-2228 ([Ala1, Thr3, Tyr4, Arg5, and Ser17]-hG-CSF) and derivative A ([Ala1, Thr3, Tyr4, Arg5]-hG-CSF) are easily crystallized and they show similar in vitro activity. On the other hand, neither rhG-CSF nor derivative B ([Ser17]-hG-CSF) are crystallized under the same 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 of hG-CSF.
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Japan.
M
M
Yamasaki
N
N
Konishi
K
K
Yamaguchi
S
S
Itoh
Y
Y
Yokoo
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000302:isolation & purification; D001681:Biological Assay; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003124:Colorimetry; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008970:Molecular Weight; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D017421:Sequence Analysis
D000596:Amino Acids; D000970:Antineoplastic Agents; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; C072115:nartograstim