Vector-derived expression of recombinant rat interleukin-6.
Rat interleukin-6 (IL-6) cDNA, coding for an important inflammation- and immune-regulatory polypeptide cytokine, was cloned into the novel expression vector pH6EX3 which directs the synthesis of inserted genes as a fusion protein with histidine hexapeptide (HH). The resultant vector (pRIL6.992) was shown to produce significant amounts of recombinant rat IL-6 fusion protein with HH at its N-terminus in various strains of Escherichia coli. The expression of the HH-IL-6 fusion protein was demonstrated to be under the control of the tac promoter and could be induced by IPTG. This protein was isolated from bacterial inclusion bodies and purified to homogeneity in a one-step procedure by affinity chromatography using a nickel-chelating column. The HH-IL-6 fusion protein isolated in this manner was biologically active as determined by hepatocyte stimulation and B9 hybridoma growth assays. Further, this activity was neutralized with a polyclonal antiserum raised against rat IL-6 protein generated in a novel fashion from rabbits infected with a recombinant human type-5 adenovirus vector expressing rat IL-6 protein (Ad5E3rIL6). The recombinant HH-IL-6 protein was then used to boost Ad5E3rIL6-immunized rabbits. This resulting antiserum was shown to neutralize recombinant and natural rat and murine IL-6 bioactivity in vitro and was useful in Western blot analysis and immunohistochemistry of rat IL-6.
Braciak
T A
TA
Department of Pathology, Molecular Virology and Immunology Program, McMaster University, Hamilton, Ontario, Canada.
Northemann
W
W
Chong
D K
DK
Schroeder
J A
JA
Gauldie
J
J
D000260:Adenoviruses, Human / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D010957:Plasmids / Q000235:genetics*; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014170:Transformation, Genetic
D018076:DNA, Complementary; D015850:Interleukin-6; D011993:Recombinant Fusion Proteins