Molecular characterization and prokaryotic expression of buffalo (Bubalus bubalis) Interleukin-6.
The study describes the characterization of Interleukin-6 cDNA and essential promoter sequences of the Indian Water Buffalo (Bubalus bubalis) and expression of the recombinant IL-6 in Escherichia coli. Buffalo IL-6 shows very high nucleotide level identity of the cDNA (98.7%) and promoter (98%) sequences with the corresponding cattle sequences. All the major regulatory elements of IL-6 promoter like AP-1, Multiple Response Element, NF-IL6, ETS binding domain and NF-kappaB binding sites show absolute conservation. Basal level IL-6 mRNA is detected in organs like liver, lung and spleen. Concanavalin A stimulated splenocytes produced maximum IL-6 mRNA at 8h poststimulation. Recombinant IL-6 production in JM109 (DE3) and BL21 (DE3) pLysS bacterial system is substantially enhanced by supplementation of rare codon tRNAs through co-transformation with a second plasmid. BL21 (DE3) pLysS strain is a more efficient producer of the IL-6 as it expressed two-fold more protein than by JM109 (DE3) cells. The study shows high-level conservation of IL-6 regulatory and coding sequences between cattle and buffalo, and indicates the use of a common reagent for studying the effects of this cytokine in these species.
Premraj
Avinash
A
Animal Biotechnology Laboratory, Rajiv Gandhi Center for Biotechnology (RGCB), Jagathy, Thycaud PO, Trivandrum 695014, Kerala, India.
Sreekumar
E
E
Nautiyal
Binita
B
Rasool
T J
TJ
D000818:Animals; D001483:Base Sequence; D002020:Buffaloes / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000502:physiology*; D015850:Interleukin-6 / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000502:physiology; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D020218:Response Elements / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics
D015850:Interleukin-6; D011994:Recombinant Proteins; D014157:Transcription Factors