Expression, purification, and characterization of recombinant human interleukin-13 from NS-O cells.
Interleukin-13 is a cytokine which is secreted by activated T lymphocytes and primarily impacts monocytes, macrophages, and B cells. A synthetic gene coding for human interleukin-13 has been prepared and cloned into expression vector pEE12. The construct was transfected into NS-O cells, which showed stable expression of the recombinant protein. A four-step purification procedure consisting of S-Sepharose, Q-Sepharose, hydroxyapatite, and Sephacryl-100 chromatographies yielded bioactive interleukin-13 of > 98% purity. The purified protein was structurally characterized. The extinction coefficient at 280 nm was determined to be 5678 M-1 cm-1. Amino acid sequencing confirmed that the N-terminus of the purified protein was intact. Electrospray mass spectrometric analysis, size-exclusion chromatography, and SDS-PAGE revealed that the biologically active protein is monomeric and unglycosylated. Mass spectrometry and a chemical assay for free sulfhydryls indicated that the four cysteine residues of interleukin-13 are involved in two intramolecular disulfide bonds. The circular dichroism spectrum confirms that interleukin-13 belongs to the alpha-helical family of cytokines. A biologically inactive covalent trimer also forms in the cell culture, but can be separated from the monomer by the hydroxyapatite and size-exclusion chromatographies. These data indicate that human interleukin-13 retains many structural similarities to human interleukin-4, from which it arose by a gene duplication event.
Cannon-Carlson
S
S
Department of Biotechnology Development, Schering-Plough Research Institute, Union, New Jersey 07083, USA.
Varnerin
J
J
Tsarbopoulos
A
A
Jenh
C H
CH
Cox
M A
MA
Chou
C C
CC
Connelly
N
N
Zavodny
P
P
Tang
J C
JC
D001483:Base Sequence; D002460:Cell Line; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation / Q000235:genetics*; D006801:Humans; D018793:Interleukin-13 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D013058:Mass Spectrometry; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000302:isolation & purification
D017931:DNA Primers; D018793:Interleukin-13; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds