High yields of interleukin-8 produced by a synthetic gene expressed in Escherichia coli and purified with a single antibody affinity column.
We developed a highly efficient expression system for the production of interleukin-8 (IL-8) in Escherichia coli. A synthetic gene used in the vector was designed to code for the 72-amino-acid form of IL-8 and incorporate additional new restriction sites. IL-8 was expressed in very large amounts in the periplasmic space and extracted by a gentle method which did not utilize denaturants. About 69% of the protein extracted from the periplasmic space was properly processed IL-8. A single anti-IL-8 monoclonal antibody affinity chromatography column yielded homogeneous IL-8 as determined by HPLC molecular sieve chromatography and amino-terminal sequencing. Between 14 and 22 mg of IL-8 was purified per liter of bacterial culture, in which the wet weight of E. coli was 7.6 g/liter. The recombinant IL-8 was fully active compared to published data and a commercially available preparation of recombinant IL-8. Our IL-8 and the commercial product had identical neutrophil binding isotherms, chemotactic activities, and enzyme release properties.
Miller
E J
EJ
Department of Biochemistry, University of Texas Health Center, Tyler 75710, USA.
Cohen
A B
AB
Carr
F K
FK
Hayashi
S
S
Chiu
C Y
CY
Lee-Ng
C T
CT
Mullenbach
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D001681:Biological Assay; D002634:Chemotaxis, Leukocyte; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D006801:Humans; D016209:Interleukin-8 / Q000031:analogs & derivatives / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D017421:Sequence Analysis
D016209:Interleukin-8; D011994:Recombinant Proteins