2021.06.02.4
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INVESTIGATION / RESEARCH
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Soluble production of a full-length human papillomavirus type 16 L1 protein by Escherichia coli

Yunier Serrano1, Susana Miraidys Brito, Elsa Pimienta1, Alina Falero1,
Karen Marrero1
Available from: http://dx.doi.org/10.21931/RB/2021.06.02.4 (registering DOI)
ABSTRACT
Persistent infection with human papillomavirus type 16 (HPV16) causes
the development of cervical cancer. Escherichia coli is a cost-effective host
successfully used to develop a second-generation vaccine against HPV, based on
the purification of soluble truncated L1 protein variants. Previous attempts to
produce soluble full-length HPV16-L1 protein by E. coli have failed. This study was aimed at cloning a Cuban
HPV16-L1 gene in E. coli and assessing
its expression as a soluble full-length L1 protein by manipulating culture
conditions. The L1 gene
was amplified from a Cuban patient’s cervical sample and cloned into pET28a and
pBAD/Myc-HisA
vectors. Production and solubility of L1 protein were evaluated in E. coli TOP10
harboring pBADHPV16-L1 plasmid and E.
coli BL21-(DE3), Rosetta-(DE3)/pLysS, and SHuffle® T7 Express lysY strains harboring
pETHPV16-L1 plasmid, grown under arabinose (0.2%)- or isopropyl
β-D-1-thiogalactopyranoside (IPTG, 100 µM)-induction or Super Broth-based auto-induction
for 24 and 48 h. The recombinant plasmids pETHPV16-L1
and pBADHPV16-L1 were constructed. The HPV16-L1 protein was produced
insoluble to high levels in conventionally IPTG-induced E. coli-pETHPV16-L1 cells. However,
under auto-induction, soluble full-length HPV16-L1 protein was successfully produced
at similar levels by E. coli BL21
(DE3), Rosetta (DE3) pLysS and SHuffle®
T7 Express lysY cells, reaching up
to 7.2 ± 0.5% and 14.3 ± 1.6% of the total
proteins in the soluble fraction after growing for 24 and 48 h, respectively. It
is concluded that the auto-induction procedure at
18 °C with 30 µM IPTG and 100 rev/min promotes soluble full-length HPV16-L1 protein
production by E. coli.
Keywords: Human papillomavirus type 16, Escherichia
coli, L1 protein
INTRODUCTION
Human papillomavirus (HPV) are non-enveloped double-stranded DNA viruses 1, of which more than 200 types have been described as pathogens in humans 2. HPVs
are classified into high-risk (H) and low risk (LR) according to their
oncogenic potential 2. The persistent infection of approximately 15
high-risk HPV types causes almost all cervical cancer cases (CC) and immediate
precursor lesions 3. Of these genotypes, HPV16 and HPV18 account for approximately 70% of global CC cases 4. As a whole, the
CC is the third most common carcinoma among women 5. In particular, HPV16
is the most prevalent high-risk genotype globally 6,7, and studies
have shown that it is related to a large proportion of other HPV-associated
cancers worldwide 7.
The papillomaviruses consist of L1 and L2 structural proteins, being
L1 protein the preferred target for HPV vaccine development due to its self-assembling
into virus-like particles (VLPs) mimic the structure of native virions 4,8.
The L1-VLPs retain the vast majority of the natural virus’s neutralizing
epitopes and can induce high titers of neutralizing antibodies 4. There
are currently three HPV-VLP-based prophylactic vaccines available: Cervarix,
Gardasil-4, and Gardasil-9 3. Clinical trials have shown that these vaccines
induced neutralizing and protective antibodies to prevent HPV-associated infections
7. However, the vaccines’ high production and delivery costs are
significant barriers to global implementation, mainly in low-incoming countries
where cervical cancer results in higher mortality 9.
In Cuba, the CC constitutes the fourth cause of cancer deaths in women 10,
being the HPV16 the most frequently found genotype 5. The molecular epidemiologic results
support the national implementation of vaccination against HPV in our country 5,11, unfeasible at present due to the high costs of current
vaccines. Thus, there is a pressing need for more cost-effective
vaccines 1.
The bacterium Escherichia coli
constitutes the most attractive host for recombinant protein production due to
their low cost, high productivity, and rapid production rates 12, 13.
Using this host, a VLP-based vaccine was recently licensed in China 14.
L1 proteins in this vaccine (including HPV16-L1 protein) were produced in
truncated form and purified from E. coli
soluble fraction 3,15. However, soluble full-length HPV16-L1 protein
production in E. coli has not yet been
achieved since L1 protein is mainly detected in the cells induced by the cells’
insoluble fractions IPTG 16. The auto-induction culture is a strategy
that allows accumulating soluble recombinant protein in E. coli cytoplasm 17-19, as well as the employ of low
temperature and low inductor concentration 19-21. Therefore, it
would be attractive to produce high amounts of soluble full-length HPV16-L1
protein, considering that L1 protein purification from soluble fraction may be
a cost-effective alternative since refolding of purified protein is avoided 3.
Thus, the present study aimed to assess soluble full-length L1 protein production from a wild type HPV16-L1
gene in E. coli by manipulating
culture conditions.
MATERIALS AND METHODS
Reagents, E. coli Strains, and Plasmids
Restriction enzymes, T4 DNA ligase and Pfu DNA polymerase, PCR reagents,
1kb DNA Step Ladder, Broad Range Protein Molecular Weight Markers, Wizard® Minipreps
DNA purification system, and Wizard® Gel and PCR Clean-Up System were purchased
from Promega (USA). Agar, tryptone, yeast extract, and sodium chloride were
from OXOID (England). Kanamycin, ampicillin, and chloramphenicol antibiotics were
from AppliChem (Germany). IPTG, the auto-induction medium components, and all
chemicals used in SDS-PAGE and Western Blot
were from Merck (Germany). The E.
coli strains and plasmids used in this study are listed in Table 1.

Table 1:
Bacteria and plasmids used in this study.
All DNA manipulations, including restriction digestions, ligations, and
agarose gel electrophoresis, were performed according to standard procedures 24.
Preparation of E. coli competent cells
and plasmid transformation protocols were adapted from Li et al.
25.
Amplification of HPV16-L1
gene and cloning into pUC18NotI
plasmid
The HPV16-L1 gene (1527 bp) was amplified by Polymerase Chain
Reaction (PCR) using total DNA isolated from an HPV16-diagnosed biopsy sample from
a Cuban patient 26 and
the primer pairs 5’- CCATGGGTCTTTGGCTGCCTAGTG -3’ and 5’- AGATCTCTTACAGCTTACGTTTTTTGCGTT
-3’. The amplified gene was flanked by NcoI and BglII sites (underlined) at the
5’ and 3’ ends, respectively, and it was cloned
into HincII-digested pUC18NotI vector
generating the pUCHPV16-L1 plasmid in E.
coli Mach1. Restriction
analysis and nucleotide sequencing (Macrogen, Republic of Korea) were used to confirm
the HPV16-L1 gene presence in recombinant clones.
Construction of E. coli HPV16-L1 expression plasmids
The wild type HPV16-L1 gene (1518 bp) encoding full-length L1protein from pUCHPV16-L1 was sub-cloned into pET28a (+) and pBAD/Myc-HisA expression vectors as a
NcoI-BglII fragment, obtaining the pETHPV16-L1 and pBADHPV16-L1
recombinant plasmids, respectively. These plasmids were characterized by
restriction, then pBADHPV16-L1 was introduced into E. coli TOP10, while the pETHPV16-L1 plasmid was transformed into E. coli BL21(DE3), Rosetta (DE3)pLysS,
and SHuffle® T7 Express lysY.
pBAD/Myc-HisA and pET28a(+) vectors were introduced into E. coli TOP10 and E. coli
BL21(DE3), respectively, as expression controls.
SDS-PAGE and Western Blot analysis
For determination of molecular weight, identity testing, and solubility of HPV16-L1 protein, 10% (w/v) sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (10 % SDS-PAGE) under
reduced conditions 27 was used, followed by staining with Coomassie
brilliant blue R250. For Western Blot,
all samples were diluted 1:20, except the E.
coli TOP10 lysates and electroblotted into a nitrocellulose membrane
(Sigma, USA). The HPV16-L1 protein was detected with the MAB885 (CamVir-1) antibody
(1:50 000, Merck, Germany). Blots were then treated with goat anti-mouse IgG
peroxidase (HP) conjugated (1:64 000, Cuba) and revealed with 3, 3’-
diaminobenzidine tetrahydrochloride substrate (Sigma, Germany). The intensity of
protein bands from the SDS-PAGE gel was calculated using ImageJ software 19.
Evaluation of HPV16-L1
protein production by E. coli BL21 (DE3)
and TOP 10 in test tubes
E. coli TOP10 cells harboring pBAD/Myc-HisA or pBADHPV16-L1 and E. coli BL21(DE3) cells harboring pET28a
(+) or pETHPV16-L1 plasmids were inoculated into 5 mL Super Broth medium (SB) (32 g/L of tryptone, 24 g/L of yeast extract, 5 g/L of NaCl, pH 7.2) containing appropriate antibiotics
and incubated at 37°C and 200 rev/min for 16 h. These overnight cultures were diluted 100 times in test tubes
containing 5 ml of fresh SB medium. The cells were incubated at 37°C until they reached an optical
density at 600 nm (OD600 nm) of 0.4–0.6. Then, the expression of L1
gene was induced by the addition of 0.1 mM IPTG or 0.2% (w/v) of
L(+)-arabinose. After incubation for an additional 3 h
at 37 °C, cells were harvested by centrifugation and suspended in 1x PBS (137
mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4,
pH 7.4) at an OD600nm = 6.5. The 80 μL of cell suspension was
immediately mixed with 20μl of SDS-sample buffer (0.25 M Tris-HCl, pH 6.8, 2%
SDS, 5% 2-mercaptoethanol, and 5% glycerol) and boiled at 95 °C for 10 min.
Evaluation of HPV16-L1
protein production by E. coli under conventional
induction with IPTG in shake flasks
For L1 protein production by E.
coli BL21(DE3), Rosetta, and SHuffle containing
pETHPV16-L1 plasmid, a single colony from each strain was grown overnight in 5
ml of SB medium-plus kanamycin (50 µg/ml) at 30 °C and 200 rev/min. Overnight
cultures were diluted 100 times in fresh SB
medium and incubated at 27°C
and 200 rev/min until they reached an OD600=0.4-0.6. E.
coli cells were then induced with 0.1 mM IPTG for 4 h under the same conditions. E.
coli BL21 (DE3) harboring pET28a(+) vector was included as the negative
control. Sample preparation was carried out as before for HPV16-L1 protein
production in test tubes.
Evaluation of HPV16-L1
protein production by E. coli under auto-induction
in shake flasks
Production of L1 protein by E.
coli BL21(DE3), Rosetta, and Shuffle strains
carrying the pETHPV16-L1 plasmid and E.
coli BL21 (DE3) strain harboring pET28a(+) vector were also assayed in an SB-based
auto-induction medium 28, using 30 μM IPTG instead of lactose
as auto-inducer. Thus, a single colony of
each strain was pre-cultured overnight into test tubes containing 5 ml of SB medium
with kanamycin (50 µg/ml) at 30 °C and 200 rev/min for 16 h. Then, 2 ml of the
grown-culture were transferred into a 2 L- flask
containing 200 ml of auto-induction medium supplemented with the same
antibiotic. The cultures were incubated at 18 °C and 100 rev/min for 24 or 48h.
Sample preparation was carried out as before for HPV16-L1 protein production in
test tubes.
Evaluation of HPV16-L1
protein solubility in E. coli under
different culture conditions
For assessing HPV16-L1 protein solubility from
conventionally-induced cells or auto-induced cells; E. coli BL21(DE3), Rosetta, and SHuffle
harboring pETHPV16-L1 plasmid were harvested by centrifugation at 10 000
rev/min, 10 min, 4°C (5810R, Eppendorf AG, Germany), washed twice with 50 mL of
1 x PBS and suspended in 40 ml of 1 x PBS. Then, bacterial cells were lysed
using an Emulsiflex C3 cell disrupter (Avastin, Germany), and 80 µL of each
cell lysate was saved as the whole cell’s protein (WCP). The samples’ remainder
was centrifuged (10,000 rev/min, 10 min) to separate soluble fractions (SF).
Bioinformatic tools
Gene Runner 3.05 Version was used for PCR primer design, and vector
NTI Suite 7 was employed for plasmids restriction analyses.
Statistical
analysis
All experiments were performed at
least three times. Determination of statistical differences was performed with
Prism 6 (Graphpad Software, Inc., San Diego, CA, USA) using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. Error bars presented
as the means ±
standard deviation (S.D.).
RESULTS
Cloning of HPV16-L1 gene and
construction of pETHPV16-L1 and pBADHPV16-L1 expression plasmids
PCR amplification using
total DNA isolated from a Cuban patient’s cervical sample showed a fragment of ~1.5
kb (Figure 1A), close to the expected value (1527 bp). The amplified L1 gene
codes for a 57 kDa full-length HPV16-L1 protein. The amplified fragment was
then cloned into the pUC18NotI vector
obtaining the pUCHPV16-L1 recombinant plasmid, which was subsequently sequenced.
Analysis of cloned-L1 gene nucleotide sequence by Blastn showed it was 99%
identical to the L1 gene from the HPV16 reference strain (accession number:
K027118). Nucleotide sequencing identified guanine (G) at 604 positions instead
of cytosine (C) in the L1 gene of the HPV16 reference strain, thus resulting in
a change of CAT codon histidine-202 in the L1 gene of HPV16 reference strain by
GAT codon for aspartate-202 in the cloned L1 gene. The second GGT codon, coding
for glycine-2, was identified in the cloned L1 gene resulting from the insertion
of the NcoI site required for subsequent
sub-cloning into pET28a (+) and pBADMyc-HisA expression vectors.
After HPV16-L1 gene sequencing, it was subcloned into pBAD/Myc-HisA and
pET28a(+) vectors obtaining the pBADHPV16-L1 and pETHPV16-L1 expression
plasmids. L1 presence in recombinant plasmids was verified by double
restriction with NcoI and HindIII enzymes and by single restriction with BamHI
enzyme. Said digestions yielded DNA
fragments with sizes close to the theoretical ones and showed that L1 gene was
correctly inserted under araBAD (Figure
1 B, lanes 2-3) and T7/lac promoter control
(Figure 1 C, lanes 2-3).

Figure 1. A) PCR
product analyses. Lane M: 1Kb DNA Step ladder (Promega), 1: Amplified wild-type
HPV16-L1 gene. B) pBADHPV16-L1 plasmid restriction analyses. Lane 1: undigested
pBADHPV16-L1 plasmid, lane 2: NcoI-HindIII digested pBADHPV16-L1 plasmid, lane
3: BamHI digested pBADHPV16-L1 plasmid. C) pETHPV16-L1 plasmid restriction
analyses. Lane 1: undigested pETHPV16-L1
plasmid, lane 2: NcoI-HindIII digested pETHPV16-L1 plasmid, lane 3: BamHI
digested pETHPV16-L1 plasmid.
HPV16-L1 protein
production by E. coli BL21 (DE3) and
TOP 10 strains
The IPTG-induced E. coli BL21 (DE3)/pETHPV16-L1 cells
produced HPV16-L1 protein as an intense band of about
57 kDa (Figure 2A, lane
2), which was recognized, along with other smaller species, by the HPV16-L1 specific monoclonal
antibody CamVir (Figure 2B, lane 2). In contrast,
arabinose-induced E. coli TOP 10 cells produced low levels of HPV16-L1
protein (Figure 2A, lane 4), only detected by Western blotting with CamVir
antibody (Figure 2B, lane 4).

Figure 2. HPV16-L1 protein production by E. coli from pBAD/Myc-HisA or
pET28a-derived plasmids. A): 10% (w/v) SDS-PAGE and B): Western Blot with CamVir-1 antibody of IPTG- or
arabinose-induced-cells. Lane M: Broad Range Molecular Weight Marker (Promega),
lane 1 and 2: E. coli BL21(DE3)
harboring pET28a(+) and pETHPV16-L1 plasmids, respectively. Lane 3 and 4: E. coli TOP10 harboring pBAD/Myc-HisA and
pBADHPV16-L1 plasmids, respectively.
HPV16-L1 protein production
by IPTG-induced or auto-induced-grown E.
coli BL21 (DE3), Rosetta, and SHuffle strains in shake flasks
HPV16-L1
protein production was evaluated in BL21 (DE3), Rosetta, and Shuffle
strain transformed with pETHPV16-L1 plasmid under IPTG auto-induction. E. coli BL21 (DE3), Rosetta, and SHuffle cells produced HPV16-L1 protein as
an intense band of about 57 kDa
under IPTG-induction (Figure 3A, lanes 2, 3, and 4) as
well as under auto-induction for 24 h (Figure 3D; lanes 2, 3 and 4) and 48 h
(Figure 3G; lanes 2, 3 and 4). The overproduced protein under all tested
culture conditions, as well as smaller species, reacted with the HPV16-L1 specific
CamVir antibody (Figure 3B, E and H; lanes 2, 3 and 4).
The BL21(DE3) lysates carrying the pET28a (+) vector showed a weak band (Figure 3A, D
and G: lane 2) that CamVir did not
recognize (Figure 3B, E and H: lane 2).
Conventionally-IPTG-induced cells produced similar amounts of L1
protein (Figure 3C). However, under auto-induction conditions for 24 h, BL21(DE3) produced the highest
amount of L1 protein concerning Rosetta (p<0.0001) and SHuffle (Figure
3F; p<0.01). For 48 h,
BL21(DE3) also produced the highest amount of L1 protein compared to Rosetta (p<0.0001) and SHuffle (Figure 3I; p<0.05). The L1 protein level was higher in SHuffle regarding Rosetta at 48h (Figure 3I; p<0.01).

Figure 3: Production of HPV16-L1 protein by
E. coli BL21(DE3), Rosetta, and SHuffle strains harboring pETHPV16-L1 plasmid grown under different conditions. A, D, and G: 10 % ( w/v) SDS-PAGE of
4h-IPTG-induced, 24h-auto-induced, and 48h-auto-induced cells, respectively. B,
E, and H: Western Blot with CamVir -1
antibody (1: 50 000) of 4h-IPTG-induced, 24h-auto-induced, and 48h-auto-induced
cells, respectively. Lane M: Broad Range Protein Molecular Weight Markers (Promega), lane 1: BL21(DE3)/pET28a(+) vector, Lane 2: BL21(DE3)/pETHPV16-L1,
Lane 3: Rosetta /pETHPV16-L1, Lane 4: SHuffle /pETHPV16-L1. Arrows indicate L1 protein. C, F
and I: Densitometric analyses of SDS-PAGE scanned gels. Statistical analyses
were performed by one-way ANOVA followed by Tukey’s multiple comparisons test.
Significant differences were considered at P< 0.05, P < 0.01 and
P<0.0001. The data are presented as means ± SD (N = 3). BL: BL21(DE3), Ro: Rosetta, and Sh: SHuffle.
Solubility of HPV16-L1
protein produced by IPTG-induced- or auto-induced-grown E. coli BL21 (DE3), Rosetta, and SHuffle strains in shake
flasks
The HPV16-L1
protein’s accumulation was undetected in the soluble fractions of the conventionally-IPTG-induced
cells from the three E. coli evaluated strains (Figure 4A and B: lanes 2, 4,
and 6). By contrast, in auto-induced cells, the soluble L1 protein was detected
(Figure. 4 D and G: lanes 2, 4, and 6) at similar levels by the three E. coli strains (Figure 4F and I). The percentage of HPV16-L1 protein concerning the total proteins in auto-induced
cells during 24 and 48 h was 7.2 ± 0.5% and 14.3 ± 1.6%, respectively. Moreover, part of L1 protein was produced insoluble in BL21
auto-induced for 24 h (Figure 4 D and E: lanes 1 and 2), as well as in
BL21(DE3) and SHuffle auto-induced for
48 h (Figure 4G and H: lanes 1,2,5 and 6). However, in Rosetta grown for 24 h
or 48 h, practically all the produced L1 protein accumulated in the soluble
fraction (Figure 4 D and G: lanes 3 and 4).

Figure 4: Solubility of HPV16-L1 protein
in E. coli BL21(DE3), Rosetta, and SHuffle strains harboring pETHPV16-L1 plasmid grown under different conditions.
A, D, and G: 10 % SDS-PAGE of 4h-IPTG-induced, 24h-auto-induced, and 48h-auto-induced
cells, respectively. B, E, and H: Western
Blot with CamVir-1 antibody (1: 50 000) of 4h-IPTG-induced, 24h-auto-induced,
and 48h-auto-induced cells, respectively. Lane 1: BL21(DE3) whole cell lysate,
2: BL21(DE3) soluble fraction, 3: Rosetta whole cell lysate, 4: Rosetta soluble
fraction, 5: SHuffle whole cell lysate, 6: SHuffle soluble fraction. Arrows indicate L1
protein. * Whole Cell Protein (WCP), Soluble Fraction (SF). C, F and I: Densitometric
analyses of SDS-PAGE scanned gels. Soluble HPV16-L1 protein percent among
different strains was not different, by one-way ANOVA followed by the Tukey’s
multi comparisons test. Data presented as means ± SD (N = 3). BL: BL21(DE3), Ro: Rosetta, and Sh: SHuffle.
DISCUSSION
The effectiveness of
commercially available anti-HPV VLPs-based vaccines has promoted a considerable
interest in developing vaccine candidates from cheaper hosts 8,29. VLP-based
vaccines derived from E. coli are
more cost-effective than those prepared from insect cells or yeasts at an industrial
scale. (1) A VLP-based vaccine assembled
from soluble N-terminal truncated variants of L1 protein in E. coli was developed 3, 15 and recently licensed in China 14,
showing this host’s potential for producing L1 proteins. In the present
study, a wild-type full-length HPV16-L1 gene was amplified by PCR from total
DNA extracted from a biopsy sample of an HPV16-diagnosed patient with cervical
cancer amplified gene was cloned and verified by sequencing. The cloned HPV16-L1
gene matches the European variant (Accession K02718) with the highest sequence
homology level (99 %). There was only one nucleotide difference between the
K02718 (C) and the cloned sequence (G) at position 604, changing the codon CAT
for histidine-202 in the reference sequence to GAT codon encoding aspartate-202
in the amplified product, which otherwise is required for VLPs assembly in
recombinant systems 30.
Successful expression and solubility
of recombinant proteins depend on, among the main factors: the choice of the expression
vector, expression host, medium composition, and induction conditions 13,
20. Thus, it was assessed the production level and solubility of
full-length HPV16-L1 protein in E. coli
TOP 10 transformed with the pBADHPV16-L1 plasmid and E. coli BL21(DE3), Rosetta and SHuffle transformed
with pETHPV16-L1 plasmid by conventional induction with L(+)-Arabinose
or IPTG, as well as SB-based auto-induction. L1
protein was efficiently produced in BL21(DE3) induced
with IPTG employing the pET vector series
previously used to produce the HPV16-L1 protein in E. coli host by other researchers 31. The smaller
species recognized by CamVir-1 antibody in BL21(DE3) cell
lysates agrees with previous reports on HPV16-L1 protein fragmentation from
wild-type L1 gene expressed in insect cells 30, Saccharomyces cerevisiae
32 and E. coli cells 8,31.
In contrast to BL21 (DE3)/pETHPV16-L1, the E. coli TOP10/pBAD
expression system did not efficiently produce L1 protein. The arabinose-regulated promoter has
been widely used for protein expression in E.
coli 18 to promote high amounts of soluble recombinant proteins 33.
However, in our study, L1 protein was barely detected by Western Blot,
which could be related to L1 gene expression’s lesser efficiency from the pBAD
vector series. Our results showed that the produced protein corresponds to the
L1 protein from HPV16 and that the BL21(DE3)/pET expression system is better
than TOP10/pBAD system for the production of L1 protein from the wild-type HPV16-L1
gene.
Once we verified the production of the
L1 protein by BL21(DE3) /pET expression system, we proceeded to assay its production in E.
coli BL21(DE3), Rosetta, and SHuffle carrying
the pETHPV16-L1 plasmid under induction with IPTG or
SB-based auto-induction. The results obtained showed that the HPV16-L1
protein was produced at similar levels in conventionally-induced cells. In contrast, the L1 protein production
levels in auto-induced cells were significantly different. BL21(DE3) produced the highest amount
of HPV16-L1 protein, followed by SHuffle and Rosetta. These
results are consistent with Zarschler et al., who found a slightly higher
production of single-domain antibodies in BL21(DE3) than SHuffle using the pET
system after induced with IPTG 34. The behavior in the production
levels of HPV16-L1 protein in SHuffle and Rosetta
concerning BL21 (DE3) may be related to constitutive lysozyme production and accumulation
within cells. The lysozyme is a natural inhibitor of T7 RNA polymerase (T7 RNAP)
20, which is supplied by pLysS plasmid (10-12 copies per cell) 23 in Rosetta 35 or by miniF plasmid (1-2 copies per cell) 36 in SHuffle 37. It is
possible that, during long time-culture (like auto-induction) of
lysozyme expressing cells, the lysozyme produced reaches high levels
and decreases the T7 RNAP availability, affecting the L1 protein yield in those
cells.
The solubility of L1 protein produced
by conventionally-induced cells or auto- induced
cells was also analyzed. The soluble HPV-L1 protein production might
be advantageous for developing a successful and efficient E. coli-based HPV-LI VLPs vaccine 3, due to the soluble protein
may preserve the protective conformational epitopes 3,15.
In this regard, soluble full-length L1 protein has not been
detected by SDS-PAGE from IPTG-induced E.
coli cell 31. Likewise, in our study,
the L1 protein was found insoluble in conventionally-induced cells, which differs from the soluble production of HPV11-L1 protein from a
wild-type L1 gene using the pET vector series 38. By contrast, HPV16-L1 protein solubility
was enhanced in auto-induced cells, which is in line with previous reports about using
auto-induction as a strategy to promote soluble recombinant protein production in
E. coli 17-19. This strategy successfully
increased the solubility of recombinant proteins produced insoluble under IPTG-induction
in E. coli, such as
human tissue plasminogen (tPA)17,28 and streptococcal cysteine protease 39. Long et al. reported soluble production of full-length tPA
in E. coli, showing that auto-induction is a reliable strategy to
reach at least 50% of tPA expression in the soluble fraction E. coli 17. Three years
later, Fathi-Roudsari et al. optimized
the expression conditions for the tPA protein (truncated variant) E. coli by decreasing temperature and
using an SB-based auto-induction medium, resulting in at least three times
higher levels of active reteplase production in oxidative cytoplasm of Rosetta-gami 19. Like Fathi-Roudsari et al., we used an SB-based
auto-induction medium and 18 ⁰C grown temperature instead
of using lactose; a low IPTG concentration (30 µM) was used, which previously
has been used efficiently in auto-induction 40. Our results are also consistent with the positive effect of decreasing inducer’s
concentration and induction temperature on the recombinant protein solubility
in the E. coli cytoplasm 19-21. Thus, a
decrease in temperature during induction causes a reduction in the recombinant
protein synthesis rate and a reduction in hydrophobic interactions and consequently prevents the
formation of inclusion bodies 20. Fathi-Roudsari et al. found that decreasing
the temperature to 25 and 18
°C increased active reteplase level by 20 and 60%,
respectively 19.
On the
other hand, the amount of L1 protein accumulated in
BL21(DE3), Rosetta, and SHuffle’s soluble fraction was similar, suggesting that soluble L1 protein
production depends more on culture conditions than the genetic
background of each strain (Table 1) 20,23,35. Gu et al. produced soluble truncated HPV16-L1 protein with a purity of about 10% under
conventional induction with IPTG 41,
a level similar to those achieved in the current study (7.2 and 14 % 24 and 48 h
of autoinduction, respectively).
Our results demonstrated that a slow expression procedure that combines
growth at a low temperature, a low IPTG concentration, and a low agitation
speed under auto-induction conditions promotes soluble HPV16-L1
protein accumulation. These constitute the first report describing the successful
production of
soluble full-length HPV16-L1 protein from a wild-type L1 gene in E. coli using an auto-induction strategy. This could be valuable for using
prokaryotic auto-induction expression systems to produce HPV16-L1 antigen for
vaccine development, omitting the in
vitro refolding step for its purification. Purification of the soluble
full-length
HPV16-L1 protein for structural
and immunogenicity analyses is currently underway.
In short, a
Cuban wild type full-length HPV16-L1 gene was successfully cloned and expressed
in E. coli BL21(DE3), Rosetta (DE3)
pLysS, and SHuffle® T7 Express lysY
employing the pET system. E. coli
culture under auto-induction at 18°C, with 30µM IPTG and a low agitation (100
rev/min) promotes soluble production of full-length HPV16-L1 protein. development
of this bacterial system could be used for purifying HPV16-L1 protein from
soluble fraction and evaluating its capacity to form VLPs, which could
be used as vaccine candidate antigen.
CONCLUSION
The auto-induction procedure at 18 °C with 30 µM IPTG and 100
rev/min promotes soluble full-length HPV16-L1 protein production by E. coli.
Acknowledgment
We thank Dr.
Juan Carlos Piña for providing us with the genomic DNA for HPV16L1 gene
amplification.
Conflict of Interest
The authors declare that they have no conflict
of interest.
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Received:
28 November 2020
Accepted:
10 January 2021
Yunier Serrano1, Susana Miraidys Brito, Elsa Pimienta1, Alina Falero1,
Karen Marrero1
1Unit for Biological Products Research, Division of Research,
Development & Innovation, National Center for Scientific Research, Avenue
25 and 158. Cubanacán, Playa, La
Habana, Cuba. P.O. Box 6414.
*Corresponding
author: Yunier Serrano
National Center for Scientific
Research, La Havana, Cuba
e-mail: yunier.serrano@cnic.edu.cu;
Tel: 53-7-2086548