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com/aut
ISSN: 0891-6934 (print), 1607-842X (electronic)
Autoimmunity, Early Online: 18
! 2015 Informa UK Ltd. DOI: 10.3109/08916934.2015.1022164
Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran, 2Division of Genetics, Biology
Department, Faculty of Sciences, University of Isfahan, Isfahan, Iran, 3Department of Medical Biotechnology, Shahid Beheshti University of Medical
Sciences, Tehran, Iran, and 4Genetics & Molecular Biology Department, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Abstract
Keywords
Interferon b (IFNb) is the most important drug that has been used frequently for multiple
sclerosis treatment. This study has tried to improve the IFNb production by introducing
mutations in the coding region of IFN, while its amino acid sequence is intact. Two
recombinant vectors IFNK and IFN K+CRID were designed by site-directed mutagenesis. The
IFNK and IFNK+CRID have two substitutions in Kozak sequence and four substitutions in CRID
sequence, respectively. The Chinese hamster ovary (CHO) cell codon usage optimization was
also performed for both of them. They were transiently transfected to CHO-dhfr cell line using
Lipofectamine kit (Invitrogen, Grand Island, NY). The amount of mRNA and protein was
determined by real time PCR and ELISA. The results of this study indicate that the amount of
IFNb protein produced by CHO cells containing IFN K has been elevated up to 3.5-fold. On the
other hand, enormous amounts of IFNb mRNA and protein were produced by cells containing
IFNK+CRID construct; more than 4.6-fold and 6-fold, respectively. It could be concluded that
disruption of AT pattern in CRID element increase RNA and protein production, improve IFNb
mRNA stability and, may also enhance mRNA half-life. In a similar way, more proteins are
produced by modification of Kozak sequence.
Introduction
Interferon b (IFNb)- is the member of type I interferons that
are structurally and functionally related proteins [1]. IFNb is a
spherical 22 kDa glycoprotein with five helices and 166
amino acid residue in its structure [2,3]. All types of
interferon I bind to common cell surface receptors. Binding
of IFNb to its receptor initiate an intracellular cascade of
signaling pathways that leads to expression of IFNb-inducible
genes [4]. So, the products of these genes cause diverse
effects, including anti-viral, anti-proliferative, anti-inflammatory, immunomodulatory and other biological activities [57].
Based on these properties, IFNb has been well studied for its
clinical benefits and applications. In particular, it is a famous
drug for multiple sclerosis (MS) treatment [810]. Besides
MS, the IFNb is widely used for treatment of viral infection,
HIV-related disease, hepatitis C and also other diseases [11].
The recombinants human IFNb, Betaseron (rHuIFNb-1b)
and Avonex (rHuIFNb-1a) are approved by FDA for
MS treatments [12,13]. The rHuIFNb-1b is produced as a
Correspondence: Zohreh Hojati, Division of Genetics, Biology
Department, Faculty of Sciences, University of Isfahan, Isfahan, Iran.
Tel: +98(0)311-7932478. Fax: +98(0)311-793 2456. E-mail: z.hojati@
sci.ui.ac.ir
History
Received 13 September 2014
Revised 9 January 2015
Accepted 14 February 2015
Published online 23 March 2015
M. Kay et al.
Figure 1. Destabilizing elements in IFNb mRNA structure. There are two destabilizing (ARE) that are located in the (30 -UTR) and the coding region
(CRID). The Kozak sequence of IFNb mRNA is different in two locations in comparison with the conserve sequence which can reduce translation
efficiency. The origin of translation is indicated by a curved arrow.
Figure 2. The structural illustration of the
recombinant IFNbs. (A) The wild-type IFNb
structure that is used as a control case. (B)
Recombinant IFNb with improved Kozak
sequence. (C) Recombinant IFNb with four
point mutations in CRID region. All these
constructs were cloned in pSVM vector.
DOI: 10.3109/08916934.2015.1022164
Figure 3. Site-directed mutagenesis in Kozak sequence of IFNb by SOEing PCR. (A) Schematic diagram of site-directed mutagenesis in Kozak
sequence using SOEing PCR. (B) The PCR products of the first (line 1) and second (line 2) PCR which produce a 400-bp and a 1097-bp product,
respectively in order to make two point mutations in Kozak sequence. (C) The final PCR reaction produced a 1497-bp IFNb with two point mutations in
Kozak sequence. (D) The sequencing of IFN K that proves site-directed mutagenesis and illustrating incorporation of two point mutations in Kozak
sequence. Numbers of the fragments are in base pair.
Table 1. Oligonucleotide primer sequences for amplification of different varieties of IFN and Eef11 genes.
Name
IFN K
FP1
RP2
FP3
RP4
FP5
IFN K+CRID
RP6
FP7
RP8
IFN R
F
R
Eef11
F
R
Sequence
Purpose
50 -TCAGCCTGTTGATGAAGTAGAAG-30
50 -CTACTTCATCAACAGGCTGACAG-30
50 -GGAATTCTAACTTTATGATGAGAGAA-30
50 -TGGTATCACTATTGACATC-30
50 -TATCTCTTCTGGCTGTAA-30
50 -GACAGGATGAACTTTGAC-30
50 -ATAGACATTAGCCAGGAG-30
M. Kay et al.
Average
DCt
DDCt
2DDCt
IFNK
IFNK H
IFNK+CRID
IFNK+CRID H
IFNW
IFNW H
21.42
18.205
14.98
13.7
22.96
19.45
3.215
0.295
1.226885
1.28
2.23
4.69134
3.51
The CHO-dhfr monolayer cells were dispersed by trypsinEDTA 48 h after transfection. Cell suspensions were centrifuged at 200 g for 10 min. Total RNA was then extracted
from the cell pellet by RNeasy mini Kits (Qiagen , Hilden,
Germany) following its recommended protocol. RNA extraction was also performed from non-transfected CHO cells as a
control for native IFN expression. RNA quantity was
confirmed by spectrophotometer. The expected 18S and 28S
bands were seen by electrophoresis in a 2% agarose gel with
ethidium bromide staining. Afterward, 2 mg purified RNA was
used as the template for cDNA synthesis using the RevertAid
First Strand cDNA Synthesis Kit (Fermentas) in the presence
of hexameric random primers. The RNA and cDNA samples
were stored at 80 C.
Results
DOI: 10.3109/08916934.2015.1022164
M. Kay et al.
Figure 5. The amplification curve and Ct value of IFN and EIF1 genes in IFNK and IFNCRID samples. (A) The Ct value of IFN expression in
IFNCRID sample. The lower Ct of IFN indicates its high copy number in comparison with IFN expression in IFN K. (B) The Ct value of IFN
expression in IFNK sample. The Ct value of IFN in this case is higher than IFN expression in IFNCRID. (C) PCR products after real time PCR. Two
specific bands for IFN (177 bp) and EIF1 (297 bp) were detected and no primer-dimer formation was seen.
Discussion
The anti-inflammatory and immunomodulatory effects of
IFNb drug have proven for relapsing-remitting MS (RRMS)
treatment. The IFNb-1a and IFNb-1b are the two famous
formulations of IFNb that approved by FDA and widely used
DOI: 10.3109/08916934.2015.1022164
transfected to the CHO cell line and the RNA and protein
expressions were analyzed by real time PCR and ELISA,
respectively. The biological activity of the recombinant
interferons were also studied here. The RNA and protein
expression analyses of non-transfected CHO cells (negative
control) indicated that there was no native expression of
human IFNb in CHO cells and all the expressed IFNbs belong
to the transfected constructs. This is the main reason for
selecting this cell line in this study. The previous studies have
shown that there are two destabilizing elements in the 30 -UTR
and the CRID sequence of IFN gene [26]. Raj and Pitha in
1993 have shown that replacement of the IFNb 30 -UTR with
stable SV40 30 -UTR destabilized the transcript lesser than the
wild-type IFNb, whereas replacement of both 30 -UTR and
CRID sequence (with pBR322 and SV40 30 -UTR, respectively) made the transcripts very stable [18]. Here, for the first
time, the Kozak sequence was modified in such a way that
makes it completely similar to the conserve one. This
construct was transfected to the CHO cell line in order to
elevate the translation efficiency. The CRID element was
selected as the second target for site-directed mutagenesis.
The structure of this unstable element was disrupted by
introducing four specific point mutations in this region by
site-directed mutagenesis. IFNbK+CRID was then designed and
cloned again for the first time in this study. The real time and
ELISA analyses have proven that these mutations enhance
RNA and protein production more than 4.6-fold and 6-fold,
respectively. For efficient expression of IFN gene, the
recombinant gene with the preferred codon usage of CHO was
designed. There is no change in amino acid sequence due to
these mutations and therefore the structure of IFNb protein is
intact. Therefore, it should have its native biological activity,
like the wild-type b interferon. However, the bioactivity of the
recombinant IFNb was also confirmed by using KAC1201 kit,
which is specified for biological active IFNb. Disruption of
AT pattern in CRID element increases RNA and protein
productions, improves IFNb mRNA stability and, may
enhance mRNA half-life within the cells. Kozak sequence
has important roles in the initiation of translation and the
power of translation is somehow dependent on it. This
sequence is not matched exactly in various mRNA and often
has different residues in some positions in comparison with
the consensus one. To improve the expression levels, it may
be useful to change the original sequence in such a way as to
make it similar to the Kozak consensus sequence [19]. The
IFN K construct has two mutations in -2 and -5 position of
Kozak sequence to make it like the conserve residues. The
real time analyses indicated that this mutation has no
significant effects on the amount of RNA level in the cell,
as it has improved the amount of RNA just up to 20% in
comparison with the wild-type. When the translation rate of
mRNA is elevated, this would bring more ribosomes to IFNb
mRNA and therefore increases its stability. So, this is the
main reason for just this 20% elevation in the amount of
mRNA. According to the role of Kozak sequence in just
protein expression and translation, this result was entirely
appropriate. On the other hand, more than 3.5-fold elevation
in IFNb protein expression were detected in cells containing
IFN K, as illustrated by ELIZA analysis. Therefore, this
mutation facilitates the formation of translational complex
Declaration of interest
The authors report no conflicts of interest.
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