Int. J. Life. Sci. Scienti. Res.,
4(4):
1915-1924,
July 2018
Expression and Purification of Nisin in Escherichia coli
Huynh
Thi Xuan Mai1,
Nguyen Van Hau1, Nguyen Hieu Nghia1,
Dang Thi Phuong Thao1*
1Department
of Molecular and Environmental Biotechnology, Faculty of Biology-Biotechnology,
University of Science, Vietnam National University Ho Chi Minh City, Vietnam
*Address
for Correspondence: Dang
Thi Phuong Thao, Head,
Department of Molecular and Environmental Biotechnology, University of Science,
Vietnam National University in Ho Chi Minh City 227 Nguyen Van Cu Street,
District 5, Ho Chi Minh City, Vietnam
ABSTRACT- Fusion expression is a promising
strategy for the production bioactive peptides in Escherichia coli to enhance either soluble protein level or
purification potential. Nisin is the bacteriocin that
had been extensively studied and had been widely applied in many areas such as
food, pharmaceutical. However, scientific reports on recombinant nisin
production in E. coli are still
insufficient. In this study, we constructed a new expression plasmid containing
the coding sequence of NusA, hexahistidine
and Lactobacillus lactic nisin coding
sequence. Next, we introduced the expression plasmid into BL21 E. coli and produced the recombinant
fusion nisin in E. coli. Recombinant E. coli extract was purified by
nickel affinity chromatography and resulted in 77% yield with 55% purity. The
bioactive nisin was successfully released from NusA‑6xHis‑Nisin
fusion protein by the endonuclease. The nisin showed
its antibacterial activity on Listeria
monocytogenes with activity unit of 18.9 AU/mg. The nisin bioactivity is
stable at the temperature range of 30-90oC and in pH range of 1-12. The
results showed that the new construction was appropriate for production of
nisin bioactive peptides.
Keywords:
Nisin, Recombinant protein, E. coli,
Expression system, Bacteriocin
INTRODUCTION- Nisin is a bacteriocin which was well known and widely used in many
types of applications. Up to now, seven natural Nisin variants (A, Z, F, Q, U,
U2, H) have been recognized [1]. Nisin A and Z were the most variant
nisins in nature. Sequences of nisin A and Z was
differed in only one amino acid residue at position 27 (nisin Z contains asparagine, nisin Acontains histidine); meanwhile, nisin Q and nisin A was differed in
four residues [2]. These nisins shared
similar antibacterial spectrum. Besides, a new study showed that nisin Q can
inhibit oxidation better than nisin A [3].
Nisin is a cation
peptide from bacteria Lactococcus lactis
[4-7]. Mature nisin peptide sequence has 34 amino acids, five ring
structures in the molecule (A, B, C, D, E) with one lanthionine
(ring A) and four β methyllanthionine (rings B,
C, D, E) [1]. Hsu et. al. [8]
showed that two thio-ether rings at N-terminal of nisin
performed an important role in interacting with lipid II which was a component
involved in the formation of gram-positive bacterial peptidoglycan
cell wall. Nisin had wide antibacterial spectrum against gram-positive bacteria
and some gram-negative bacteria. Nisin inhibited the growth of those species by
forming pores in the bacterial cell membrane or inhibited the formation of peptidoglycan wall via interacting with lipid II. Thereby,
the causes of target bacterial death were cytoplasmic
membrane depolarization, ion exchange disorder, cell energy production
decreasing [5,9-11]. Furthermore, antibacterial activity of nisin
was stable in low pH or high temperature conditions [5,12].
Accordingly, nisin has been applied in food preservation and medical purposes
for almost 30 years. Nisin was approved as a safe food preservative by FAO,
FDA, and was licensed to be used in more than 60 countries [13].
The
applicability and safety of nisin brought huge needs of production. However,
production of nisin was still limited. Up to now, nisin was only produced by
natural nisin production strains.
Precursors of nisin were synthesized as a peptide of 57 amino acids.
These pre-peptide sequences need several transformation stages after the
translation to form active nisin sequences with 34 amino acids. This process required 11 components involving in prepeptide (NisA), modification (NisB and NisC), secretion (NisT), processing (NisP),
regulation (NisR and NisK)
and immunity (NisF, NisE, NisG and NisI) [14-22].
In
the face of that reality, very few successful studies of recombinant nisin
production were reported. Karakas et. al.
[23],
expressed nisin A as a fusion protein with 6xHis tag to facilitate nisin
collecting and purifying. In that research, a precursor nisin fused with 6xHis
tag in N-terminal (prenisin‑His6) was expressed in E. coli and purified using nickel
affinity column under denaturing condition [23]. However, results of
the collection of active nisin have not been reported. With the aim of
producing recombinant nisin, in this study, we constructed and expressed nisin
in E. coli in fusing form with 6xHis
and NusA tag.
MATERIALS AND METHODS- This study
proceeded in July 2017 at Department of Molecular and Environmental
Biotechnology, Faculty of Biology-Biotechnology, University of Science, Vietnam
National University Ho Chi Minh City, Vietnam.
Nisin fusion expressing vector- Nisin
coding gene (kN)
was designed according to previously published peptide sequences (BAC145) [2,3]. Gene kN was amplified by PCR reaction with specific primers
containing the recognition sites of the restriction enzymes Xhol
at the 3’ end and BamHI at the 5’ end (5’BamHI-kN). Recombinant plasmid pET43.1a‑kN was
structured by cohesive cloning with two restriction enzymes Xhol
and BamHI. DH5α clones
containing recombinant vector was selected by ampicillin
antibiotics (100µg/ml) and PCR
using T7 terminator and 5’ BamHI‑kN
primers. pET43.1a‑kN vector
was extracted and the fusion gene was analyzed by sequencing.
Inducing expression and collecting of the fusion
nisin- The plasmid pET‑kN was
transformed into E. coli BL21 (DE3) competent cells, cultured and selected in medium
containing ampicillin (100µg/ml) and PCR with primer pair 5’
BamHI‑kN/T7 terminator. Single bacterial colony E. coli BL21
(DE3)/pET‑kN
were cultured in LB medium containing ampicillin (100µg/ml), at 37oC,
250 rpm overnight. 1/20 (v/v) of the seed culture was inoculated into LB medium
containing ampicillin and continuously incubated to
log phase (OD600 approx. 0.8). IPTG was added at a final concentration of 0.8 mM and the mixture was cultured for an additional 4h at 37oC,
250 rpm. The bacterial cells
were harvested by centrifugation (5000 rpm, 5 min) and the bacterial pellet was
resuspended in lysis buffer
(Na2HPO4
50 mM, NaCl 300 mM, Imidazole 10 mM pH 7.4).
This was followed by sonication at 4oC and centrifuged (13,000 rpm, 10 min). The protein
present in supernatant and pellet were tested by SDS-PAGE.
SDS-PAGE and Western blot- The protein expression purity levels were
ascertained by SDS-PAGE, silver staining or Coomassie
blue R250 staining. The presence of recombinant proteins was determined by
Western blotting using a primary anti-6xHis-tag antibody (Invitrogen)
and a secondary anti-Ig-G antibody conjugated to HRP
(Invitrogen).
Nisin purification- The E. coli BL21
(DE3) cell extract was collected and purified by using Ni-NTA resin column (Histrap HP 1ml, GE Healthcare). Histrap HP 1 ml column was equilibrated with cell lysis solution. The supernatant was collected from cell lysis solution (Na2HPO4 50 mM, NaCl 300 mM,
Imidazole 10 mM pH 7.4).
The supernatant of cell lysis was added into column.
Non-specific binding proteins were washed with 10 times column volume by
followed buffers: Na2HPO4 50 mM,
NaCl 300 mM, Imidazole 20 mM pH 7.4. Fusion
nisin was eluted by these buffers: Na2HPO4 50 mM, NaCl 300 mM,
Imidazole 100 mM pH 7.4.
The purity of the protein was confirmed by SDS-PAGE, silver staining. The
concentrations of purified proteins were measured using Bradford assay and stored
at -30oC.
After NiNTA
purification, fusion NusA-6xHis-Nisin was then dialyzed using PBS buffer (4oC, overnight). Protein
dialyzed sample was concentrated using amicon 10kDa (Merck Millipore). The fusion nisin was
then treated by enterokinase (Department of Molecular
and Environmental Biotechnology, University of Science) at 25oC in 16hto release
fusion tag.
Evaluation of nisin bioactivity- Nisin
bioactivity was determined with indicator bacteria Listeria
monocytogeneseither by agar diffusion method or polyacrylamide
gel. Single bacterial colony Listeria monocytogenes were
cultured in TSB medium at 37oC, 250
rpm overnight. 1/20 (v/v) of the
seed culture was inoculated into TSB medium and continuously incubated at 37oC under shaking condition 250 rpm until cell density reaches 0.1 (OD600=0.1). The cultured was then diluted
with TSA 0.8% medium at1/50 (v/v). Using
agar diffusion method, the TSA 0.8% diluted cultured were poured onto a TSA
1.5% gel plate. 6 mm in diameter and 3 mm in height wells were made on the
surface of the gel and nisin samples were added into that wells. The diluted
cultured were also poured directly onto a previously run polyacrylamide
gel. The Nisin protein samples were treated with non-reducing conditions and
separated by electrophoresis. The plates and gels were then incubated at 37oC for 6h and observed for fade rings
formation.
The activity of nisin samples was
evaluated by agar diffusion method with serial double dilution. Antibacterial
activity of the samples was determined by the diameter of their inhibition
zones. The highest dilution fold (n) with inhibition diameter ≥2 mm was recorded [24].
Nisin activity unit is the reciprocal using following formula:
Activity
units (AU/ml)= 2n X 1000/ V
n =Highest dilution fold, V =Test volume
Specific
activity (AU/mg)=
Activity
units (Au/ml)/ Target peptide concentration (mg/ml)
Stability of fusion nisin- Stability
of nisin segments whose activity confirmed was tested by varying temperature
and pH. Samples were incubated at 30, 50, 60, 70, 80, 90, 100şC for 15 minutes then determined for antibacterial
activity against Listeria monocytogenes by agar diffusion method. The
pH-stability of nisin was also analyzed. Different samples of nisin were
subjected to different pH conditions from 1 to 12 at 4oC for 1h then determined for antibacterial
activity against Listeria monocytogenes by agar diffusion method.
RESULTS
Construction of expression vector for nisin in E. coli- Nisin is naturally synthesized in Lactococcus
lactis, which was well known AT rich organism [5,6,9]. Analysis of codon usage of Lactococcus lactis indicated that A and/or T ending codons are predominant in the organism [25]. For
expressing nisin in E. coli, we analyzed nisin coding sequence to address whether the sequence was adaptable to E. coli
codon usage. By using GenScript software, our result showed that nisin
coding sequence has no tandem rare codon (the CFD, Codon usage Frequency
Determination, value is 0%) in comparison to E. coli codon usage (Table 1). Besides, CAI (Codon Adaptation Index) value was 0.95 compared
to ideal value range from 0.8-1.0. Previous research demonstrated that the base
usage, codon usage and amino acid usage are changed
with GC content in linear correlation [26]. Our gene analysis data
showed GC content of nisin coding gene was 48.56% compared to the ideal range
from 30% to 70% (Table 1). The nisin gene was adaptable and can be expressed in
E. coli without optimization.
Table
1: Adaptation of nisin coding gene in
Escherichia coli
Essential
parameters
|
Actual Value
|
Ideal Value
|
CAI
|
0.95
|
0.8 – 1.0
|
CFD (%)
|
0
|
< 30
|
GC content (%)
|
48.56
|
30 – 70
|
CAI: codon adaptation index;
CFD: Codon usage frequency determination
NusA (N utilization substance A) was a
transcription termination,
which has been well known as a functional sequence that helps to increase soluble protein in E. Coli [27-29].
In the aim of expressing nisin in E. coli as soluble
peptide, we fused nisin encoding gene with NusA at N‑terminal. A 6xHis sequence was also added for purification purpose. In order to separate the
NusA-6xHis peptide from nisin peptide by enterokinase, we fused an enterokinase cutting site onto the 5’ end of nisin coding gene (Fig. 1A). The optimized nisin coding (kN) was inserted
into a pET43.1a vector by cohesive cloning with two restriction enzymes BamHI 5’ end and Xhol 3’ end. pET43.1a‑kN recombinant
vector was selected and confirmed by PCR with specific primers 5’ BamHI‑kN and T7 terminator (Fig. 1A).
Fig. 1:
Nisin expression vector construction
A: Nisin
expression vector map; B: Result of selection recombination vector by PCR with
5’ BamHI‑kN and T7 terminator; 1-5:
selective clones; 6: PCR negative control with distilled water; 7: Negative
control with pET43.1a
DNA
sequencing results confirmed the pET43.1a‑kN recombinant vector
structure was the same as the original design; kN gene was cloned in-frame into pET43.1a vector (Fig. 2).
Fig. 2:
pET43.1a-kN sequencing
Expression,
collection and purification of nisin- E. coli BL21 (DE3) was a well-known host strain for expressing recombinant protein such as: fast high-density cultivation, inactivate protease
genes and containing hsd S (rB-
, mB- ) which help to maintain
plasmid in the E. coli cell [30,31]. In order to express nisin peptides in E. coli, the E. coli BL21(DE3) was used as host strain. The recombinant strain E. coli BL21 (DE3)/pET43.1a‑kN then
were induced by 0.8 mM IPTG and analyzed. Our data on analyzing the
induced E. coli extract exerteda band of
protein at about 66kDa as expected while
that of non-induced E. coli BL21(DE3)/pET43.1a‑kNand the control E. coli BL21(DE3)/ pET43.1a did not exert the protein band (Fig. 3A). The expressed fusion protein was confirmed by
WB with anti-6xHis antibody (Fig. 3B).
Fig. 3: NusA-6xHis-Nisin fusion protein
expression in BL21 E. coli
A: Glycine SDS‑PAGE,
1. BL21 (DE3)/pET43.1a; 2. BL21(DE3)/pET43.1a/IPTG
cell disruption; 3. BL21(DE3)/pET43.1a/IPTG
pellets; 4. BL21(DE3)/pET43.1a/IPTG supernatants;
5. BL21(DE3)/pET43.1a-kN; 6. BL21(DE3)/pET43.1a-kN/IPTG cell disruption;
7. BL21(DE3)/pET43.1a-kN/IPTG pellets; 8.
BL21(DE3)/pET43.1a-kN/IPTG supernatants;
9. Protein ladder
B: Western
blot with 6xHis antibodies, 1. BL21(DE3)/pET43.1a; 2. BL21(DE3)/pET43.1a/IPTG cell
disruption; 3. BL21(DE3)/pET43.1a/IPTG pellets;
4. BL21(DE3)/pET43.1a/IPTG supernatants;
5. BL21(DE3)/pET43.1a-kN;
6. BL21(DE3)/pET43.1a-kN/IPTG cell disruption; 7. BL21(DE3)/pET43.1a-kN/IPTG pellets; 8. BL21(DE3)/pET43.1a-kN/IPTG supernatants; 9. Protein
ladder
NusA‑6xHis-Nisin
was
purified and concentrated using affinity chromatography with NTA resin column (Fig.
4). Nisin collected from
elution phase showed the purity of 55%. The fractions were
then dialyzed and concentrated using a
10 kDa
cut-off amicon.
NusA-6xHis-Nisin was finally collected with 65% in purification (Table 2).
Table 2:
NusA-6xHis-Nisin purification
Purification steps
|
Target protein content (µg)
|
Purity (%)
|
Yield (%)
|
Cell lysis
|
4436,02
|
20,8
|
100
|
Ni-NTA
|
3423,13
|
55,00
|
77,17
|
Amicon filtration
|
2950,31
|
65,00
|
66,71
|
Fig. 4: NusA-6xHis-Nisin purification
1. Protein ladder; 2. BL21 (DE3)/pET43.1a-kN/IPTG
cell disruption; 3. Flow phase;
4. Wash
phase; 5. Elution phase; 6.Concentrated fusion nisin
Antibacterial activity of recombinant nisin- Recombinant nisin was cleaved from
NusA-6xHis-Nisin fusion protein and analyzed for anti Listeria monocytogenes activity. Our results showed that cleaved
nisin was observed on SDS-PAGE as a band at 3.5 kDain
size (Fig. 5A) with strong antibacterial activity (Fig. 5B). Furthermore, agar
diffusion assay showed that the bacterial indicator was inhibited after
treatment with recombinant nisin in compare to NusA-6xHis-Nisin and ampicillin (50 µg/ml). A clear 3mm inhibition zone was
observed while NusA-6xHis-Nisin did not deliver observable effect (Fig. 5C).
Our result of antibacterial assay strongly indicated that recombinant nisin
produced by this study inhibits the Listeria
monocytogenes. Consequence, recombinant nisin was diluted and tested by diffusion
assay. We successfully collected nisin peptide with enterokinase
treatment. The highest dilution fold was 2 (n=1) and antibacterial activity was
18.9 AU/mg.
Fig. 5: Antibacterial activity of recombinant
nisin.
A: Tricine SDS‑PAGE
fusion nisin after enterokinase treatment
B: Evaluation antibacterial activity of
recombinant nisin on gel polyacrylamide
C: Evaluation antibacterial activity of recombinant
nisin by agar diffusion method
1. Ampicillin (50µg/ml); 2. NusA-6xHis-Nisin; 3. Recombinant
nisin
D:
Specific activity of recombinant Nisin
Stability of recombinant nisin- Stability was an important aspect
that makes nisin become widely applicable in many areas [12,32].
Stability of our recombinant nisin was evaluated with different pH and
temperature conditions. Nisin were incubated at 30, 50, 60, 70, 80, 90, 100, and
121şC then subjected to
antibacterial assay. The result showed that nisin bioactivity was stable at
temperature from 30 to 90oC. Nisin bioactivity significantly
decreased at higher temperatures and become inactivated at 121oC (Fig. 6A). Recombinant
nisin was also treated in different pH conditions (pH1-12) for 1 hour and
analyzed for antibacterial activity. The experimental results demonstrated that
recombinant nisin is stable in pH range of 1-12 (Fig. 6B).
Fig. 6: Heat and pH stability of recombinant nisin
A: Heat stability of recombinant nisin
B: pH stability of recombinant nisin
DISCUSSION- Basic nisin, an antibacterial polypeptide, was
originally identified from Lactococcus lactis [4-7]. Owning a demand for large
amounts of nisin in different applications, high-level expression and
purification of the bacteriocin are interested to
attract many scientists. Their studies focused on two main directions:
improvement and optimization the culture conditions for nisin Lactococcus lactic strains culture and fermentation
or produce nisin as recombinant protein [23,33-42]. For producing a
recombinant nisin, some hosts such as E. coli,
Saccharomyces
cerevisiae, Lactococcus
lactis MG1363 have been used [23,33,43]. In
this study, BL21 E. coli strain was used for the production recombinant nisin. E. coli
expression system owns some advantages in producing heterologous
recombinant protein such as cost effectiveness, time saving, easy culture, fast
growth, and easy recovery of the recombinant protein [30,31,44].
Previous studies showed the result of expressing nisin in E. coli
but its bioactivity [23,45]. In this study, a new strategy to produce active recombinant nisin
using E. coli expression system was introduced. This expression method was succeeded
to archive recombinant nisin protein purified with nickel affinity
chromatography. Our data was also demonstrated that the stable of recombinant
nisin at temperature range of 30 - 90oC
and in pH range of 1-12.
CONCLUSIONS- In summary, the study
indicates that nisin protein could be expressed functionally in E. coli by
fusing it with NusA. The productivity of
NusA-6xHis-Nisin was achieved approximately 59 mg/L of induced culture (related
to recombinant nisin with antibacterial activity 18.9 AU/mg). This result
demonstrated an effective production of biologically active nisin. It opened a
prospect of production of nisin as recombinant protein.
ACKNOWLEDGMENT- We thank to Gene
Technology and Application Group and Laboratory of Molecular Biotechnology for
great support in this study.
CONTRIBUTION OF AUTHORS
This study was designed by Dang Thi
Phuong Thao and Nguyen Hieu
Nghia. Nguyen Van Hau and
Huynh Thi Xuan Mai equally
contributed on data collection. Data analysis and interpretation for the work
were carried out by Nguyen Hieu Nghia,
Huynh Thi Xuan Mai and
Nguyen Van Hau. Dang Thi
Phuong Thao draftted the
article and Huynh Thi Xuan
Mai wrote it. The article was critically revised and approved to be published
by Dang Thi Phuong Thao.
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