April - June 2007 - Kasetsart University

Kasetsart J. (Nat. Sci.) 41 : 335 - 345 (2007)
Cloning, Expression, Purification and Biological Activities
of Recombinant Mouse Interleukin-2 in E. coli M15
Sanchai Chantajorn 1 , Ratchanee Hongprayoon 2 * and Thaweesak Songserm 3
ABSTRACT
Molecular cloning, sequencing and expression of recombinant mouse interleukin 2 (rmIL-2)
were described. The interleukin-2 (IL-2) cDNA, 450 base pairs in length with repeating CAG, was
amplified using specific primers. The IL-2 cDNA showed high homology at 100%, 100%, 91%, 96%
and 94% with five strains of mice previously reported (GeneBank accession number AY147902.1,
MMU41494, MMU41504, MMU41505 and MMU41506). The IL-2 gene was cloned into the pDrive
cloning vector and consequently expressed using pQE30 expression vector which provided high
expression level of the recombinant protein. The predicted rmIL-2 sequence is 161 amino acids with a
molecular weight of 19 kDa. The expressed protein was then purified by Ni-NTA column under denaturing
condition. Analysis of the rmIL-2 by SDS-PAGE demonstrated two bands of 19 and 38 kDa representing
monomeric and dimeric forms of this protein. The biological activity in stimulating T-cell proliferation
was also described and the binding signal to the receptor was easily observed by immunofluorescence.
Key words: mouse interleukin-2, cloning, protein expression, protein purification, immunofluorescence
INTRODUCTION
Interleukin-2 (IL-2) is a growthpromoting
activator for bone marrow-derived T
lymphocytes (Smith, 1989), and was among the
first cytokines to be characterized at the molecular
level. Interleukin-2 was the major autocrine
growth factor for T lymphocytes, and the quantity
of IL-2 synthesized by activated CD4 + T cells was
an important determinant of the magnitude of
immune response. The action of IL-2 on T-cells
was mediated by binding to IL-2 receptor proteins.
This system was perhaps the best understood
mechanism of all cytokine receptors (Morgan et
al., 1976). Interleukin-2 exerts its effects on many
cell types, the most prominent of which is the T
lymphocyte. Indeed, one of the most rapid
consequences of T cells activation through its
antigen receptor is the de novo synthesis of IL-2.
This was quickly followed by expression of a high
affinity IL-2 receptor on surface membrane of
CD4 + T cells, CD8 + T cells, B cells and natural
killer (NK) cells (Lenardo et al., 1999).
Interleukin-2 induce cells proliferation via pro-
1 Centre for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand.
2 Department of Plant Pathology, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen Campus,
Nakhon Pathom, 73140, Thailand.
3 Department of Veterinary Pathology, Faculty of Veterinary, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom
73140, Thailand.
* Corresponding author, e-mail: agrrat@ku.ac.th
Received date : 15/08/06 Accepted date : 13/02/07