Isolation and characterization of three recombinant human granulocyte colony stimulating factor His-->Gln isoforms produced in Escherichia coli.
This report demonstrates that three variant isoforms of recombinant methionyl human granulocyte colony stimulating factor are present in small quantities in the crude preparation solubilized from Escherichia coli inclusion bodies. These isoforms were separated from the main form of the factor during purification and further isolated by a series of cationic exchange chromatographic separations. They exhibit full in vitro biological activity and have slightly lower pI's. Structural characterization of the intact proteins and their isolated peptides by sequence determination and mass spectrometric analysis revealed that they are methionyl granulocyte colony stimulating factors having a His-->Gln replacement at sequence position 53, 157, or 171, respectively. The specific His-->Gln change suggests the occurrence of mistranslation during protein synthesis. These variant forms are chromatographically separable during purification and are not detectable in the final purified form of the factor.
Lu
H S
HS
Amgen, Inc., Amgen Center, Thousand Oaks, California 91320.
Fausset
P R
PR
Sotos
L S
LS
Clogston
C L
CL
Rohde
M F
MF
Stoney
K S
KS
Herman
A C
AC
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003062:Codon; D004926:Escherichia coli; D005973:Glutamine; D016179:Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D006639:Histidine; D006801:Humans; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D003062:Codon; D011993:Recombinant Fusion Proteins; D005973:Glutamine; D016179:Granulocyte Colony-Stimulating Factor; D006639:Histidine