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ISSN: 0891-6934 (print), 1607-842X (electronic)
Autoimmunity, Early Online: 18
! 2015 Informa UK Ltd. DOI: 10.3109/08916934.2015.1022164

FULL-LENGTH RESEARCH PAPER

Effects of disruption of the nucleotide pattern in CRID element


and Kozak sequence of interferon b on mRNA stability and
protein production
Maryam Kay1, Zohreh Hojati2, Maryam Heidari2, Zahra Bazi3, and Hassan Korbekandi4
1

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 IFN K and IFN K+CRID were designed by site-directed mutagenesis. The
IFN K and IFN K+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
IFN K+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.

CRID sequence, ELISA, interferon b, Kozak


sequence, real time PCR

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

nonglycosylated protein in Escherichia coli, whereas


rHuIFNb-1a is an expressed interferon in Chinese hamster
ovary (CHO) cells as a glycosylated protein with 10-fold more
activation in comparison with its previous version [14,15].
However, the elevation of the amount of the produced
interferon has been the main challenge in its production
process. The rapid degradation and transient expression of
IFNb is due to the presence of a few different varieties of
destabilizing elements on its messenger RNA (mRNA). The
IFNb mRNA degradation is induced by the diverse elements
that are located in the 30 untranslated region (30 -UTR) and
also coding region. The 30 -UTR of IFNb has an AU-rich
element (ARE), which belongs to class II. Class II AREs have
at least two overlapping nanomers UUAUUU (U/A) (UA) U
in a U-rich environment. There is another ARE destabilizing
element in the coding region, named coding region instability
determinant (CRID). These elements are recognized by some
special proteins (like 65 kDa protein) that facilitate mRNA
degradation [1618]. Beside these destabilizing elements, the
translation efficiency of eukaryotic mRNA depends on the
other factors too. Normally, the type of nucleotide sequences
surrounding the AUG codon (Kozak sequence) is the main
issue for the translational efficiencies (Figure 1). The
translation efficiency strongly depends on the similarity

M. Kay et al.

Autoimmunity, Early Online: 18

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.

degree between this region and the known consensus


sequence, as illustrated by different varieties of studies [19].
Overall, there are a lot of studies on IFNb in order to improve
its little translation and production. In this study, two novel
recombinant vectors are designed. Each vector has a specific
mutation in IFN open reading frame (ORF) in order to
improve its mRNA stability and translation. Therefore, ARE
elements in the CRID and Kozak sequence of IFN gene were
targeted by site-directed mutagenesis (Figure 2). Finally, the
enhanced amount of IFNb mRNA and its protein were
analyzed by real time polymerase chain reaction (PCR) and
ELISA methods, respectively.

Materials and methods


Plasmid construction
The human blood samples were used to amplify HuIFN . In
order to obtain IFN containing point mutations in Kozak
sequence, splicing by overlap extension PCR (SOEing PCR)
was used (Figure 3A). Two set primers were designed
according to the HuIFN gene (GenBank: M25460.1) and

synthesized by Bioneer (Table 1). Briefly, three consecutive


PCR reactions were used in order to substitute two nucleotides in IFNb Kozak sequence in such a way to make it similar
to the conserve sequence. The P1 and P4 primers were used in
the first PCR reaction. The PCR program was performed as
below: a denaturation step at 94  C for 5 min, then 15 cycles at
94  C for 1 min, 59/5  C for 30 s, 72  C for 90 s and a final
extension cycle at 72  C for 10 min. The second PCR reaction
was carried out by using the P2 and P3 primers and the
following PCR reaction. A denaturation step at 94  C for
3 min, then 15 cycles at 94  C for 1 min, 53  C for 30 s, 72  C
for 2 min and a final extension cycle at 72  C for 10 min. Both
PCR products were analyzed on 1% agarose gel and purified
by DNA extraction kit (Qiagen, Valencia, CA).
The final PCR reaction was done by using those two PCR
products and the P1 and P2 primers. The PCR program
consisted of a denaturation step at 94  C for 5 min, then 25
cycles at 94  C for 1 min, 51  C for 30 s, 72  C for 3 min and a
final extension cycle at 72  C for 10 min. The final PCR
product was gel purified, cut and ligated into pSVM vector
to produce a recombinant plasmid containing IFNb with

Kozak and CRID mutations in interferon

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 Eef1 1 genes.
Name
IFN K
FP1
RP2
FP3
RP4
FP5
IFN K+CRID
RP6
FP7
RP8
IFN R
F
R
Eef1 1
F
R

Sequence

Purpose

50 -GAA GGA CCA ACT GTA TCT TTT AGT G-30


50 -AGC TGA ATT CTA ACT TTA TGA TGA GAG-30
50 -TCG TGT TGC CAC CAT GAC-30
50 -GTC ATG GTG GCA ACA CGA-30
50 -GGAATTCTCAGGTCGTTTGC-30

Inducing Kozak mutation

Inducing CRID mutation

50 -TCAGCCTGTTGATGAAGTAGAAG-30
50 -CTACTTCATCAACAGGCTGACAG-30
50 -GGAATTCTAACTTTATGATGAGAGAA-30
50 -TGGTATCACTATTGACATC-30
50 -TATCTCTTCTGGCTGTAA-30
50 -GACAGGATGAACTTTGAC-30
50 -ATAGACATTAGCCAGGAG-30

consensus Kozak sequence (IFN K). The structure of the


recombinant vector was confirmed by sequencing
(Figure 3D).
The second construct has IFN gene with point mutations
in both Kozak and CRID sequence in order to not only
increase its mRNA stability, but also improve its protein
translation. For efficient expression of IFN gene in CHO
cells, the recombinant gene with the preferred codon usage of

Real time analyses

CHO was also designed. In this case, unstable sequence AAC


TTT TAC TTC ATT AAC AGA CTT AC have been changed
to AAC TTC TAC TTC ATC AAC AGG CTG AC by
inducing four substitution mutations using the SOEing PCR
method. In order to keep both Kozak and CRID mutations, the
PCR reactions were performed on IFN K gene. Two specific
sets of primers were designed, modified (Table 1) and used in
three successive PCR reactions to induce those target point

M. Kay et al.

mutations in CRID sequence (the procedure and details of


PCR reactions are approximately as the same as mentioned
earlier for the first SOEing PCRs). The final product was
extracted from agarose gel by DNA extraction kit (Qiagen),
digested by EcoRI enzyme and ligated into the digested
pSVM vector to produce a recombinant IFN K+CRID gene
with mutations in both Kozak and CRID sequence. The
structures of these recombinant vectors were initially confirmed by RFLP analysis and then by sequencing. The wildtype IFN gene was cloned in pSVM vector and also used as
a control in this study (this construct obtain from Medical
University of Isfahan).
Cell culture and transfection
The CHO-dhfr cells (dihydrofolate reductase-negative) are
epithelial-like and can be grown as an adherent culture. This
cell line was cultured in Dulbeccos Modified Eagle Medium
(DMEM, High Glucose, GlutaMAX (Gibco, Grand Island,
NY)) containing 10% fetal bovine serum (FBS) (Gibco, Grand
Island, NY), 1% penicillin/streptomycin (Gibco, Grand Island,
NY); supplemented with 0.1 mM hypoxanthine (Sigma),
0.016 mM thymidine (Sigma), 0.002 mM MTX (methotrexate; Sigma). The CHO cell line was grown at 37  C, 97%
humidity, 5% CO2 condition and, 24 h prior to the transfection; 1  105 cells were seeded in 12 well plates. Serum-free
media was used to transfect 500 ng plasmid in 6080%
confluent cells by using the Lipofectamine kit
(Lipofectamine LTX with Plus Reagent) according to
the manufacturers instructions. After 6 h, the medium
was exchanged by fresh media and incubated for an
additional 24 h.
RNA extraction and cDNA preparation

Autoimmunity, Early Online: 18

Table 2. 2DDCt analyses for calculating IFN gene expression.


Name

Average

DCt

DDCt

2DDCt

IFN K
IFN K H
IFN K+CRID
IFN K+CRID H
IFN W
IFN W 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

0.5 pmol/ml of each primer, 40 ng/ml cDNA and12.5 ml


Maxima SYBR Green qPCR Master Mix included PCR
buffer, MgCl2, dNTP mixture and SYBR Green . The
reaction mixture was subjected to the first denaturation step
at 95  C for 10 min, followed by 45 cycles of denaturation at
94  C for 1 min, primer annealing step at 50  C for 30 s and
primer extension at 72  C for 20 s. A final extension at 72  C
was also performed for 5 min. In order to check the
reproducibility of the results, each sample was analyzed in
triplicate (and average Ct values were used for further
analyses). The specificity of the amplified product was
confirmed by analyzing the melting curve, in which the
incubation temperature was increased from 65  C to 95  C.
The relative gene expression of IFN was calculated by the
2DDCt method (Table 2) [20]. Two standard curves were
plotted for two 10-fold serial dilutions (1000 ng to 1 ng) of
IFNb and eIF1a cDNAs after calculation of their Ct values.
The estimated R2 of the linear standard curve was40.985 that
indicated the accepted efficiency of designed primer sets. All
experiments were carried out in triplicate and the average Ct
values of these three independent experiments (repeats) were
used for further analysis.

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.

Determination of IFN concentration by ELISA

Real time PCR

Cloning of recombinant constructs

The expression of IFN genes was determined by real time


PCR, using a Fermentas kit (Maxima SYBR Green qPCR
Master Mix (2X), ROX Solution provided) according to its
manufacturer protocol. The eukaryotic initiation factor 1 a
(eIF1 ) was used as a housekeeping gene due to its
determined stable expression. Specific primers (with no
false priming sites, no formation of primer-dimer and no
secondary structures) were designed for IFN and eIF1
(Table 1) by AlleleID 7.8 and Oligo7 program. Amplification
reactions were performed in a volume of 25 ml with

The site-directed mutagenesis in the Kozak sequence was


made by the SOEing PCR method. Two sets of specific
primers were designed for amplification of two fragments 400
(using P1 and P4 primers) and 1111 bp (using P2 and P3
primers), (Figure 3B). These PCR products were purified and
used in a SOEing PCR (using P1 and P2 primers), generating
a 1511-bp fragment that contains 30 -UTR, coding region and
50 -UTR of IFN gene (Figure 3C). Two induced point
mutations in this construct have made the IFN Kozak
sequence as the same as the conserve Kozak sequence. The

IFNb protein expression was detected by HuIFNb ELISA kit


(Invitrogene , Grand Island, NY) according to the instructions that had been supplied by the manufacturers protocols.
This kit is used to determine the amount of biologically active
HuIFNb in culture medium. A standard curve was then
plotted for the serial dilutions, including 0, 50, 100, 150 and
200 (IU/ml) of standard HuIFNb. Each sample (100 ml) was
finally added to the separately designed wells and detected by
enzyme-linked immunosorbent assay (ELISA) reader at
450 nm. The HuIFNb concentrations in the samples were
worked out based on the plotted standard curve.

Results

DOI: 10.3109/08916934.2015.1022164

Kozak and CRID mutations in interferon

Figure 4. Confirmation of the structure of the


reconstructed plasmids by restriction digestion analysis. (A) The recombinant plasmids
were isolated from the transformed colonies;
columns 1, 2 and 3 represent the extracted
plasmids of IFN W, IFN K and IFN CRID,
respectively. (B) The structure of extracted
plasmids confirmed by digestion with EcoRI
enzyme. The digestion products of IFN W,
IFN K and IFN CRID are loaded in 1, 2 and
3 columns. The 1500-bp band belong to
IFN gene. Numbers are in base pair.

second recombinant construct containing point mutations in


Kozak and CRID sequence was also created by a SOEing
PCR procedure. Two 571- and 900-bp fragments were
amplified during two different sets of PCR reactions. These
PCR products were then used in the third PCR reaction in
order to produce a 1471-bp final fragment. The purified final
PCR products were digested by EcoRI enzyme and then
ligated to pSVM single cut vector (EcoRI). The plasmids
containing recombinant IFN genes were transformed in
XL1-Blue strain of E. coli. The transformed colonies were
selected by bluewhite selection and confirmed by colony
PCR. Plasmid extraction and digestion were performed by a
Fermentas kit and EcoRI enzyme, respectively. The cloning
process was confirmed by the right sizes of the digested
fragments (Figure 4). Finally, RFLP PCR and sequencing
were carried out for the molecular analysis of the structure of
these two recombinant constructs.
Transfection and RNA extraction
These three plasmids (IFN W, IFN K, IFN K+CRID) were
transiently transfected in CHO-dhfr cells by using the
Lipofectamine kit (Invitrogen). The conditions of the transfection were optimized according to the manufacturers
instructions. An equal number of the cells (6  106) were
transfected using the optimized condition (500 ng
DNA + 2.5 ml Lipofectamine + 1 ml Plus, respectively). RNA
extraction was performed by RNeasy mini Kits, using
collected trypsinized cells after 48 h. The integrity and purity
of the extracted RNA was analyzed by two different
procedures. Initially, the RNA samples were analyzed by
gel electrophoresis. The sharp and clear 28s and 18s rRNA
bands were detected. The UV spectroscopy was then used to
assess the purity of extracted RNAs. The absorbance of
diluted RNA in 10 mM TrisCl, pH 7.5 was measured at 260
and 280 nm. The ratio of A260/A280 for all purified RNA
was 1.82.1.
Analysis of IFNb m RNA by real time PCR
The cDNA of three constructs were synthesized and used as a
template for the real time PCR reactions. The recommended
concentration of primers and cDNA, by Fermentas kit

protocol, (Maxima SYBR Green qPCR Master Mix (2X),


ROX Solution provided) were sufficient for amplification of
IFN and eIF1 genes. The annealing temperature optimization was carried out by gradient PCR at a range of 4654  C.
The efficient results, with no primer-dimer and nonspecific
products, were obtained in 50  C (thus, this temperature was
selected for amplification of both IFN and eIF1 genes). All
the experiments were repeated in three times in order to show
the reproducibility of the study. The final result of real time
PCR was obtained and analyzed by the 2-DDCt method. The
real time PCR products were loaded on a 1% agarose gel and
expected fragment of IFN (177 bp) and eIF1 (297 bp)
genes detected appropriately. These results were exactly
proved the transcriptional expression of the IFNb constructs
in vitro (Figure 5C). The real time analyses of transfected
cells with three constructs (IFN W, IFN K, IFN K+CRID)
were positive, whereas non-transfected CHO cells (negative
control) has shown no native expression of IFN . The
amplification curve of these constructs were analyzed
(Figure 5A and B). The amount of IFN K+CRID mRNA was
elevated more than 4.7-fold in comparison to the IFN W. On
the other hand, the induced mutations in Kozak sequence
(IFN K) had made no significant effect on the amount of
IFN mRNA as illustrated by the results. The amount of
mRNA was elevated just 20% in this construct (Figure 6A).
Analyses of protein production by ELISA
The equal number of the CHO-dhfr cells (6  106) were
transiently transfected with the recombinant constructs. The
supernatant medium was collected to evaluate the recombinant IFNbs biological activity and protein production by
ELISA. The ELISA was performed three times for each
sample in order to prove its reproducibility. Concentration of
IFNb was determined using the standard curve and the Oneway ANOVA and Tukeys HSD Post-hoc tests were used for
statistical analyses. There was significant difference between
the protein expression of recombinants and control groups
(IFNbW). The average protein concentration of IFNbW,
IFNbK and IFNbK+CRID was 7.45, 26.07 and 44.7 (IU/ml).
This huge amount of IFNb protein (26.07 IU/ml) indicates
that the mutated Kozak sequence could increase and elevate
the translation (3.5-fold). The expression of a more stable

M. Kay et al.

Autoimmunity, Early Online: 18

Figure 5. The amplification curve and Ct value of IFN and EIF1 genes in IFN K and IFN CRID samples. (A) The Ct value of IFN expression in
IFN CRID 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 IFN K sample. The Ct value of IFN in this case is higher than IFN expression in IFN CRID. (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.

Figure 6. Comparison of IFNb mRNA and its


protein level between different varieties of
recombinant IFNbs (A) The IFN CRID construct increases IFN RNA expression more
than 4.6-fold, whereas IFN K has no significant effect on IFN expression and just
increases IFN RNA expression 20% in
comparison with the wild-type construct.
(B) The IFN CRID and IFN K constructs
increase IFN protein expression more than
6-fold and 3.5-fold, respectively.

IFNb mRNA and elevation of its translation are the main


reasons for production of such enormous amount of IFNb
protein (44.7 IU/ml, 6-fold) in CHO cells containing
IFN K+CRID constructs. Therefore, the data analyses indicate
that the IFN K and IFN K+CRID constructs increase IFNb
protein expression more than 3.5-fold and 6-fold, respectively
(p50.05) in comparison with the IFNbW (Figure 6B).
Furthermore, This HuIFN-b ELISA kit specifically determines the biologically active HuIFN-b and thus overcomes
the lack of specificity of the bioassay. So, the results indicate
the bioactivity of the recombinant HuIFN-bs, which are
produced in this study.

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

for MS treatment. The MS patients have used IFNb frequently


with the successful effects in past decades. MS is a central
nervous system related disease; the main reason of disability
in young population that affects more than 2 million people in
the world [8,9]. According to the fast growth of MS in the
world, scientists have tried to improve the IFNb quality and
quantity by using protein and genetic engineering methods.
Different studies have tried to improve IFNb solubility, halflife or reduce its immunogenicity (in order to diminish the
drug side effects) [2123]. A modified IFNb with less
immunogenicity was provided by inducing a few substitutions
at some residues of human IFNb [24]. Otto et al. in 2009 have
managed to enhance hydrophilic property of the protein
surface by a few substitutions of some specific residues with
hydrophilic amino acid (specifically, serine, tyrosine or
threonine) [25]. In this study, two recombinant constructs
were designed in order to not only improve IFNb mRNA
stability, but also enhance its translation. Two recombinant
constructs and also a positive control (IFNbW) were

Kozak and CRID mutations in interferon

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

and increases the ribosomal activity, and so elevates the level


of protein production. The effect of Kozak mutation in
IFN K+CRID constructs is also very clear. There are two major
sources for elevation in protein production in this construct,
elevation in mRNA stability (CRID; more accumulated
mRNA in the cell) and in translation (Kozak, a very rapid
initiation step). In conclusion, these precise and directed
modifications in IFNb have improved interferon beta protein
production, meanwhile its biological activity is intact. So the
result of this study may be helpful for pharmacological and
biotechnological industries to not only improving product
quality but also diminishing the drug cost.

Declaration of interest
The authors report no conflicts of interest.

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