High level expression and refolding of mouse interleukin 4 synthesized in Escherichia coli.
Mouse Interleukin 4 is a 20-kDa glycoprotein, synthesized by activated T lymphocytes and mast cells, which regulates the growth and/or differentiation of a broad spectrum of target cells of the immune system, including B and T lymphocytes, macrophages, and hematopoietic progenitor cells. Using an inducible recA promoter and the g10-L ribosome-binding site, recombinant non-glycosylated interleukin 4 (IL-4) was expressed as 17% of total cellular protein in Escherichia coli inclusion bodies, as a reduced, inactive 14.5-kDa polypeptide. The protein was refolded and aggregates dissociated when three disulfide bonds were reformed by slowly decreasing the concentration of guanidine hydrochloride and cysteine. The oxidized monomer was purified to homogeneity by sequential ion-exchange and size exclusion chromatography. When compared with native IL-4, E. coli-derived IL-4 displayed an identical specific activity of 4-7 x 10(7) units/mg. This recombinant IL-4 contained a three-amino-acid NH2-terminal extension, which did not affect its biological activity. Purified biologically active protein consisted of three isoforms as shown by two-dimensional gel electrophoresis, with a pI greater than 9.0. These data suggest that neither glycosylation nor the NH2 terminus of mouse IL-4 play a critical role in contributing to its in vitro biological activity.
Levine
A D
AD
Searle Discovery Research, Monsanto, St. Louis, Missouri 63198, USA.
Rangwala
S H
SH
Horn
N A
NA
Peel
M A
MA
Matthews
B K
BK
Leimgruber
R M
RM
Manning
J A
JA
Bishop
B F
BF
Olins
P O
PO
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D015847:Interleukin-4 / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping
D011994:Recombinant Proteins; D015847:Interleukin-4