Structural and Functional Characterization of Recombinant Interleukin-10 from Indian Major CarpLabeo rohita.
Interleukin-10, an important regulator of both the innate and adaptive immune systems, is a multifunctional major cytokine. Though it is one of the major cytokines, IL-10 from the Indian major carp,Labeo rohita,has not yet been characterized. In the present study, we report large scale production and purification of biologically active recombinant IL-10 ofL. rohita(rLrIL-10) using a heterologous expression system and its biophysical and functional characterization. High yield (~70 mg/L) of soluble rLrIL-10 was obtained at shake flask level. The rLrIL-10 was found to exist as a dimer. Far-UV CD spectroscopy showed presence of predominantly alpha helices. The tertiary structure of the purified rLrIL-10 was verified by fluorescence spectroscopy. Two-dimensional gel analysis revealed the presence of six isoforms of the rLrIL-10. The rLrIL-10 was biologically active and its administration significantly reduced serum proinflammatory cytokines, namely, interleukin 1β, TNFα, and IL-8, and augmented the NKEF transcript levels in spleen ofL. rohita. Anti-inflammatory role of the rLrIL-10 was further established by inhibition of phagocytosis using NBT reduction assayin vitro.The data indicate that the dimeric alpha helical structure and function of IL-10 ofL. rohitaas a key regulator of anti-inflammatory response have remained conserved during evolution.
Gene Regulation Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India; School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.
Sweta
S
Karan
Fish Health Management Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751 002, India.
Pujarini
P
Dash
Gene Regulation Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Himani
H
Kaushik
Fish Health Management Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751 002, India.
Pramoda K
PK
Sahoo
Gene Regulation Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Lalit C
LC
Garg
School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.
Aparna
0000-0001-9761-7768
A
Dixit
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