April - June 2007 - Kasetsart University

other strains of mice have different effects on the
biological activity of IL-2 (Matesanz and Alcina,
1996). The IL-2 cDNA did not contain the
hydrophobic leader sequence of a 20 amino acid
peptide and the expressed rmIL-2 was purified
from the cells later. According to Robb et al.
(1981), even though the IL-2 exhibited O-linked
glycosylation at threonine 3 of N-terminus and the
E. coli system did not provide the posttranslational
glycosylation, it did not affect the IL-2 activity
nor change its activity in standard bioassay. The
functional significance of glycosylation of IL-2
was not known but it was likely that it enhances
solubility in aqueous environments. Thus an Nterminal
20 amino acid sequence was reported to
be essential for the interaction with the IL-2
receptor (Eckenberg et al., 2000). The
polymorphism of the CAG sequence has been
reported among different strains of mice including
C3HeB/FeJ mouse (AY147902.1), RF mouse
(MMU41494), C57BL6/J mouse (MMU41504),
CZECHII/Ei mouse (MMU41505), and BKL
Kasetsart J. (Nat. Sci.) 41(2) 343
mouse (MMU41506) (GeneBank data base) which
contained the sequences of 8, 8, 12, 21 and 21
codons, respectively. Characterization of the rmIL-
2 by ProtParam program (ExPASy) showed that it
consisted of 149 amino acids of mature IL-2
protein and 12 amino acids of protein tag from
the expression vector. The expressed protein was
estimated to weigh 18,489 Da, with the isoelectric
point (pI) at 5.87 with good solubility. However,
the protein bands observed on the SDS-PAGE
were found to be 19 and 38 kDa which were
predicted as a monomeric and dimeric forms of
the protein. The increased molecular weight from
the data obtained by program analysis may due to
the phosphorylation of the rmIL-2 (Adachi et al.,
1997; Brennan et al., 1997; Gesbert et al., 1998;
Justement, 2001; Cook and Unger, 2002; Michelle
et al., 2003; Stoker, 2005). The rmIL-2 were
applied to the cell culture with the following
addition of XTT to examine its biological activity.
The result showed that the activity was raised
according to the increasing concentration of rmIL-
Figure 6 The binding of the recombinant mouse IL-2 to the IL-2 receptors was analyzed by
immunofluorescence. Cells stained with sulforhodamine B (SRB) illustrating red color and
the signal for IL-2 binding showed greenish fluorescence (arrows). (A) Non-stimulated cells
and (B) mitogen-stimulated cells, after 6 h of incubation.