Modified Escherichia coli B (BL21), a superior producer of plasmid DNA compared with Escherichia coli K (DH5alpha).
ABSTRACT Plasmid DNA (pDNA) is an emerging experimental vaccine, produced in E. coli, initially targeted for viral diseases. Unlike traditional protein vaccines whose average dose is micrograms, the average dose of pDNA is on the scale of milligrams. Production yields are, therefore, important for the future development of this vaccine. The E. coli strains currently used for pDNA production, JM109 and DH5alpha, are both suitable for production of stable pDNA due to the deletion of recA and endA, however, these two E. coli K strains are sensitive to growth conditions such as high glucose concentration. On the other hand E. coli BL21 is less sensitive to growth conditions than E. coli JM109 or DH5alpha, this strain grows to higher densities and due to its active glyoxylate shunt and anaplerotic pathways is not sensitive to high glucose concentration. This strain is used for recombinant protein production but not for pDNA production because of its inability to produce stable pDNA. To adapt E. coli BL21 for stable pDNA production, the strain was mutated by deleting both recA and endA, and a proper growth and production strategy was developed. Production values, reaching 2 g/L were obtained using glucose as a carbon source. The produced plasmid, which was constructed for HIV clinical study, was found to have identical properties to the plasmid currently produced by E. coli DH5alpha.
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Conference Paper: مروری بر مهندسی ژنتیک سویه های باکتریایی جهت تولید واکسن-, -; 04/2006
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ABSTRACT: The need for the development of economic high plasmid production in Escherichia coli cultures is emerging, as a result of the latest advances in DNA vaccination and gene therapy. In order to contribute to achieve that, a model describing the kinetics involved in the bioproduction of plasmid by recombinant E. coli DH5α is presented, as an attempt to understand the complex and non-linear metabolic relationships and the plasmid production occurring in dynamic batch culture environments, run under different media compositions of glucose and glycerol, that result in distinct maximum biomass growths (between 8.2 and 12.8 g DCW/L) and specific plasmid productions (between 1.1 and 7.4 mg/g DCW). The model based on mass balance equations for biomass, glucose, glycerol, acetate and plasmid accurately described different culture behaviors, either using glucose or glycerol as carbon source, or mixtures of both. From the seventeen parameters obtained after model simplification, the following ten parameters were found to be independent of the carbon source composition: the substrate affinity constants, the inhibitory constants of biomass growth on glycerol by glucose, of biomass growth on acetate by glycerol and the global biomass growth by acetate, and the yields of biomass on acetate, acetate on glucose and glycerol, and plasmid on glucose. The parameters that depend on the culture composition, and that might explain the differences found between cultures, were: maximum specific growth rates on glucose, glycerol and acetate; biomass yield on glucose and glycerol; and plasmid yield on glycerol and acetate. Moreover, a crucial role of acetate in the plasmid production was revealed by the model, with most of plasmid production being associated to the acetate consumption. The model provides meaningful insight on the E. coli dynamic cell behavior concerning the plasmid bioproduction, which might lead to important guidelines for culture optimization and process scale-up and control.Journal of Biotechnology 07/2014; · 2.88 Impact Factor
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ABSTRACT: The market development of plasmid biopharmaceuticals for gene therapy and DNA vaccination applications is critically dependent on the availability of cost-effective manufacturing processes capable of delivering large amounts of high-quality plasmid DNA (pDNA) for clinical trials and commercialization. The producer host strain used in these processes must be designed to meet the upstream and downstream processing challenges characteristic of large scale pDNA production. The goal of the present study was to investigate the effect of different glucose feeding strategies (batch and fed-batch) on the pDNA productivity of GALG20, a pgi Escherichia coli strain potentially useful in industrial fermentations, which uses the pentose phosphate pathway (PPP) as the main route for glucose metabolism. The parental strain, MG1655ΔendAΔrecA, and the common laboratory strain, DH5α, were used for comparison purposes and pVAX1GFP, a ColE1-type plasmid, was tested as a model. GALG20 produced 3-fold more pDNA (∼ 141mg/L) than MG1655ΔendAΔrecA (∼ 48mg/L) and DH5α (∼ 40mg/L) in glucose-based fed-batch fermentations. The amount of pDNA in lysates obtained from these cells was also larger for GALG20 (41%) when compared with MG1655ΔendAΔrecA (31%) and DH5α (26%). However, the final quality of pDNA preparations obtained with a process that explores precipitation, hydrophobic interaction chromatography and size exclusion was not significantly affected by strain genotype. Finally, high cell density fed-batch cultures were performed with GALG20, this time using another ColE1-type plasmid, NTC7482-41H-HA, in pre-industrial facilities using glucose and glycerol. These experiments demonstrated the ability of GALG20 to produce high pDNA yields of the order of 2100-2200mg/L.Journal of Biotechnology 07/2014; · 2.88 Impact Factor
COMMUNICATION TO THE EDITOR
Modified Escherichia coli B (BL21), a Superior
Producer of Plasmid DNA Compared With
Escherichia coli K (DH5a)
Je-Nie Phue,1Sang Jun Lee,2Loc Trinh,1Joseph Shiloach1?
1Biotechnology Core Laboratory, NIDDK NIH Bethesda, Bldg 14A Room 173,
Maryland 20892; telephone: 301-496-9719; fax: 301-451-5911; e-mail: firstname.lastname@example.org
2Lab of Molecular Biology, NCI NIH Bethesda, Maryland
Received 1 April 2008; revision received 5 May 2008; accepted 12 May 2008
Published online 29 May 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.21973
ABSTRACT: Plasmid DNA (pDNA) is an emerging experi-
mentalvaccine, producedinE.coli,initially targetedforviral
diseases. Unlike traditional protein vaccines whose average
of milligrams. Production yields are, therefore, important
for the future development of this vaccine. The E. coli strains
currently used for pDNA production, JM109 and DH5a,
are both suitable for production of stable pDNA due to
the deletion of recA and endA, however, these two E. coli K
strains are sensitive to growth conditions such as high
glucose concentration. On the other hand E. coli BL21 is
less sensitive to growth conditions than E. coli JM109 or
DH5a, this strain grows to higher densities and due to its
active glyoxylate shunt and anaplerotic pathways is not
sensitive to high glucose concentration. This strain is used
for recombinant protein production but not for pDNA
production because of its inability to produce stable pDNA.
Toadapt E. coli BL21for stablepDNA production, the strain
was mutated by deleting both recA and endA, and a proper
growth and production strategy was developed. Production
values, reaching 2 g/L were obtained using glucose as a
carbon source. The produced plasmid, which was con-
structed for HIV clinical study, was found to have identical
properties to the plasmid currently produced by E. coli
Biotechnol. Bioeng. 2008;101: 831–836.
? 2008 Wiley Periodicals, Inc.
KEYWORDS: pDNA: E. coli B; high density
Vaccines that used plasmid DNA (pDNA) as a physical
vector encoding an antigen of interest provided a promising
method for preventing diseases such as HIV-AIDS, Ebola,
malaria, and cancer (Bacon and Sedegah, 2007; Boyer et al.,
2000; Martin et al., 2006) introduced in 1990 when purified
DNA was injected intramuscular which yielded both gene
expression and biological activity (Jiao et al., 1992; Wolff
et al., 1990). Compared with protein vaccines where micro-
gram quantities of the antigen are required, milligram
amounts of pure, super coiled pDNA are needed in order to
2007). Up to 8mgDNA were given tovolunteersin a phaseI
escalation study of a multiclade HIV-1 DNA vaccine
(Graham et al., 2006).
Production of pDNA vaccines is currently being done by
transforming E. coli with the plasmid containing the gene
that encodes the antigen of interest and a promoter active in
mammalian cells (Diogo et al., 2001; Montgomery et al.,
1993). Following the growth of the bacteria and amplifica-
tion of the plasmid, often involving temperature induction
(Carnes et al., 2006; Chartain et al., 2005), the plasmid is
recovered and purified. The yields depend on the plasmid
type, its copy number, the gene size, the culture growth
conditions, and the temperature induction step (Chen et al.,
1997; Lahijani et al., 1996). E. coli strains DH5a and JM109
are currently being used for the production of pDNA
vaccines mainly because these strains are endA and recA
negative (O’Kennedy et al., 2000; Wang et al., 2001). The
endA product is endonuclease I that degrades double-
stranded DNA and, therefore, affects the stability of the
produced pDNA (Schoenfeld et al., 1995). recA functions in
ATP-dependent homologous recombination by catalyzing
DNA strand exchange reactions (Bell, 2005). The yield of
pDNA from E. coli DH5a obtained by fed batch fermen-
source is reported to be from 0.2 to 1.3 g/L (Chartain et al.,
Compared to E. coli K, DH5a or JM109 strains, E. coli
BL21 has better growth and metabolic characteristics (Phue
et al., 2005). More importantly, the strain is insensitive to
glucose concentration because of its active glyoxylate shunt,
gluconeogensis and anaplerotic pathways, and more active
TCA cycle. The outcome is lower acetate production and
Correspondence to: J. Shiloach
? 2008 Wiley Periodicals, Inc.
Biotechnology and Bioengineering, Vol. 101, No. 4, November 1, 2008 831
to metabolic stress caused by producing large amount of
recombinant proteins (Hoffmann et al., 2002; Wittmann
et al., 2007). Taking into consideration the anticipated
amount of pDNA that will be needed for its potential use
as a vaccine, it was important to explore the production
capability of E. coli B. So far, this strain has not been used
for pDNA production probably because it contains endA
and recA. Past efforts for improving production yields
concentrated on exploring growth strategies of E. coli K
strains, but not on strain optimization. In this work, we
investigate the ability of E. coli BL21 and its derivatives to
produce an HIV pDNA vaccine, comparing its productivity
and carbon source utilization to E. coli DH5a.
Materials and Methods
Bacterial Strains and Plasmids
E. coli BL21 (F?, ompT, hsdSB(rB?,mBþ), dcm, gal) and
DH5a (endA1, recA1, gyrA96, thi, hsdR17, relA1, supE44
80 lacZDM15) strains were purchased from
Promega Corp. (Madison, WI). MG1655 strain and P1 vir
phage were in the NIH laboratory collection.
Gene knock-outs were carried out by recombination
using plasmid pKD46 as previously described (Datsenko
and Wanner, 2000). Chloramphenicol and kanamycin
markers in plasmids pACYC184 and pACYC177 (New
England Biolab, Inc., Beverly, MA) were amplified by PCR
toknockoutendA andrecA genes, respectively.Sequences of
endA gene disruption primers are 50-TGCGGTGGTACT-
TATACACCGGGAAGCCCTGGGCCAACTTT and 50-AG-
recA gene inactivation primer sequences are 50-GCGGCA-
GTCAAGTCAG. PCR products carrying antibiotic markers
and homologous region (?50 bp) were electroporated into
E. coli MG1655 carrying pKD46 where lambda recombinase
was fully expressed by L-arabinose during culture at 308C.
Two hours after electroporation and incubation at 378C,
cells were spread on LB agar plates containing chlroam-
phenicol (10 mg/mL) or kanamycin (25 mg/mL) Among
candidate colonies, specific gene disruptions were identified
by PCR with primer which can hybridize upstream or
downstream of deleted endA and recA genes, respectively.
The disrupted genes, carrying the drug markers, were
transferred to a BL21 strain by standard P1 transduction
(Miller, 1992). Gene disruptions in the BL21 strain were
reconfirmed by PCR. In order to change an antibiotic
marker in pVRC5737 plasmid (kindly supplied by the
Vaccine Research Center, NIH, Bethesda, MD), bla gene
with ampicillin resistance in pUC19 was amplified using
and 50-TACCCGGGTAAACTTGGTCTGACAGTTA, digested
by ClaI and XmaI, and ligated to ClaI- and XmaI-digested
pVRC5737, finally to construct pVRC5737Ap.
Fed-batch fermentations were carried out using a 5 L
working volume fermentor (NBS, Edison, NJ) with 3.0 L of
extract,5 g/LNaCl, 5g/LK2HPO4,and glycerol orglucose as
metal solution, 0.6 g thiamine (DH5a) and glucose or
glycerol to final concentration of 5 g/L were added. The
fermentor was inoculated with 200 mL of overnight culture
and was cultivated in batch mode at 308C; pH 7, and 30%
dissolved oxygen concentration maintained by adaptive
controlled system adjusting agitation rate and air or oxygen
flow rate (Hsiao et al., 1992). After approximately 4 h (OD
600 nm?10), fed-batch modebasedon dissolved oxygen set
point of 30% was implemented by using Yokogawa model
UT152 controller and a Watson Mallow pump delivering
6 mL per dose of 50% glycerol or glucose feeding solution
containing 25% yeast extract, 10 mM MgSO4, 1 mL/mL
trace metal solution and 0.6 g thiamine (DH5a). Plasmid
DNA expression was induced by increasing the growth
temperature from 308C to 428C for 10 h.
Samples for acetic acid, glycerol, and pDNA were
collected throughout the fermentation process. The samples
were centrifuged at 14,000g for 10 min at 48C, the super-
natant was kept at ?208C for acetate analysis, and the cell
pellets were quickly frozen by dry ice and stored at ?208C
for pDNA extraction.
Acetic acid in the culture supernatant was detected using a
Boehringer Mannheim Kit 148621. Acetate determination is
based on the formation of NADH, while acetate is converted
to citrate and acetyl-CoA in the presence of acetyl-CoA
synthetase. Glucose in the culture supernatant was deter-
OH). Glycerol was determined using a glycerol assay kits
obtained from R-Biopharm, Inc. (Marshall, MI).
Quantitative and Biological Assay
For quantitative pDNA production, frozen pellet samples
equivalent to 3 OD were used and pDNA was extracted
using QIAprep spin columns(QIAGEN, Inc., Valencia, CA).
The concentration of DNA was determined by measuring
absorbance at 260 nm (A260). The purity of DNA was
determined by running a 0.8% agarose gel and measuring
the absorbance ratio at 260 and 280 nm (A260/A280). The
isolated DNA had an A260/A280ratio of 1.9–2.0. Biological
activity was done by checking the protein produced by the
Biotechnology and Bioengineering, Vol. 101, No. 4, November 1, 2008
plasmid: Transfection of the prepared plasmid produced
was done using a Profection Mammalian transfection kit
(Promega U.S. Madison, WI, USA). The transfected culture
(HEK 293T cells, Johns Hopkins, MD) were allowed to
incubate for 24 h, the cells were dislodged via a pipette and
the cells precipitated with gentle centrifugation. The cell
pellet was washed with PBS and then resuspended in 400 ml
BugBuster brand cell lysis reagent containing benzonase
(Novagen, Madison, WI). The cells supernatant was diluted
1:1 with a reducing gel buffer and 20 ml of the diluted
solution were loaded onto a 4–12% Bis Tris Mops SDS gel
(Invitrogen, Carlsbad, CA) and electrophoresed for 1 h at
200 V. The gel was blotted to a 0.22 U nitrocellulose
membrane,the blot wasblockedand probedwith rabbitanti
GP-120 overnight, after washing the blot was reacted with
the second antibody, goat anti rabbit for 1 h, washed again
and developed using the BCIP reagent.
Knocking Out endA and/or recA Genes in E. coli BL21
Initially, we tried to transform pKD46 (Datsenko and
Wanner, 2000) into BL21 strain, but failed to make a BL21
that harbors pKD46, which would be useful in direct gene
deletion. It was assumed that the pKD46 plasmid is unstable
in the BL21 strain due to endA and recA genes in the
chromosome. Therefore, a gene knock-out strategy was
changed to inactivate endA and recA genes in MG1655 strain
and to transfer those mutations to BL21 strain by P1
transduction. Recipient BL21 cells were transducted with
P1 lysates of MG1655 endA and recA strains, to make
BL21 endA and BL21 recA, respectively. P1 transductant
candidates were selected and purified on plates containing
appropriate antibiotics. To make the double knock-out
strain, P1 lysate of endA mutants was used in an attempt to
transduct recA strain, however no transductants were
obtained because P1 transduction requires RecA recombi-
nases in recipient cells. In contrast, recA and endA double
mutants were easily obtained when we transfer recA
mutation to endA BL21 strains by P1 transduction. The
mutant BL21 strains were confirmed by PCR (results not
shown). After confirmation, E. coli BL21 mutants were
transformed by plasmid pVRC5737Ap (pDNA) for DNA
Production of pDNA in E. coli DH5a
Growth of E. coli DH5a and plasmid DNA production is
shown in Table I and Figure 1. Using glycerol as carbon
pDNA production as function of the bacterial strain and the carbon source.
Strain Carbon sourceFinal cell OD Final productivity (mg/L) Maximal specific productivity (mg/g wet cell)
Growth, pDNA production and acetate accumulation, during fed batch growth of DH5a on glycerol (A) and glucose (B).
Phue et al.: Modified Escherichia coli B (BL21)
Biotechnology and Bioengineering
source, the bacteria grew to an OD of 70 at 308C; pDNA
production reached a maximum of 0.99 g/L following
temperature increase to 428C. The pDNA production and
themaximum celldensitywerelower when glucose wasused
as a carbon source (Fig. 1B), cell OD reached 100 before
decreasing to 73 toward the end of the temperature
induction period when and the pDNA production was
0.107 g/L. In contrast to the glycerol-based growth, where
acetate concentration was close to zero throughout the run,
in the glucose based growth acetate concentration reached
50 g/L even when the glucose concentration was kept below
0.5 g/L throughout the growth.
Production of pDNA in E. coli BL21 and endA?
The growth and pDNA production from E. coli BL21 and
its three derivatives (BL21 endA?, BL21 recA?, and BL21
endA?/recA?) is shown in Table I and Figure 2. The cell
growth using glycerol was similar; it reached OD close to
200, but pDNA production was different in the various
strains. It reached maximum of 1.9 g/L in BL21 recA?and
in BL21 recA?/endA?, and 0.26 and 0.19 g/L respectively in
BL21 and BL21 endA?. The specific production was six to
ten times higher in BL21 recA?and BL21 recA?/endA?
Growth, pDNA production and acetate accumulation, during fed batch growth of BL21 (A), BL21 endA?(B), BL21 recA?(C) and BL21 endA?/recA?(D) on glycerol.
Biotechnology and Bioengineering, Vol. 101, No. 4, November 1, 2008
compared with BL21 and BL21 endA?. The production was
similar when E. coli BL21 endA?/recA?grew on glucose
(instead of glycerol) as a carbon source; the bacterial
concentration reached OD of 190 and produced 1.7 g/L of
pDNA (Fig. 3, Table I), acetate concentration did not exceed
The plasmid produced by the improved producer E. coli
BL21 endA?/recA?was compared to the plasmid produced
by the DH5a strain. The analysis using agarose gel
electrophoresis (Fig. 4a) and evaluating biological activity
following gp120 expression from 293T cells (Fig. 4b),
indicatethat the plasmids produced by the two E. coli strains
Discussion and Summary
the plasmid ability to express a protein antigen which, in
turn, promotes antibody production and confers host
immunity. This approach was tested successfully for several
viral infections such as West Nile virus vaccine, licensed in
2005 by the USDA for use in horses and is currently in phase
II trial in humans (Acambis, 2006), and H5N1 influenza
DNA vaccine (Sharpe et al., 2007) currently undergoing
phase I human trials (NJAID, 2007). Effective amounts of
injected pDNA are approximately 100–1,000 times higher
than the amount of protein antigen used for vaccine, and
therefore emphasis is currently devoted to optimization of
its production. The existing production procedure is based
on growing transformed E. coli K strains (DH5a or JM109)
to high density by using glycerol as carbon source.
Optimization efforts to increase production have focused
on improving the growth strategies but have not improved
the production strain.
The central carbon metabolism in E. coli B (BL21)
operates more efficiently than in E. coli K (JM109), (Phue
et al., 2005). E. coli BL21 can tolerate high concentrations of
glucose and its growth rate andacetate accumulation arenot
influenced by the glucose concentration as is the case with
E. coli K (JM 109). it was therefore important to evaluate
the production capability of the E. coli B strain. The specific
(e) BL21 recA?(glycerol); (f) BL21 endA?/recA?(glycerol); (h) DH5a (glucose); (i) BL21 endA?/recA?(glucose). The arrow indicates the location of the super coil plasmid DNA.
B:Biologicalevaluation:Expressionofgp145asafunctionofplasmidDNAfromdifferent E.colihosts. 293Tcellsweretransfectedwith1mg/wellofplasmidDNAfromE.coliDH5a,
(lane b) 1 mg/well pDNA from E. coli endA?/recA?(lane c), and 0.83 mg/well HIV044(EnvA) as a control (lane d) Marker XP (lane a).
A: Gel electrophoresis analysis of purified pDNAs on 0.8% agarose. Lanes a and g, size marker; (b) DH5a (glycerol); (c) BL21 (glycerol); (d) BL21 endA?(glycerol);
growth of BL21 endA?/recA?on glucose as a carbon source.
Growth,pDNA production andacetate accumulation, during fedbatch
Phue et al.: Modified Escherichia coli B (BL21)
Biotechnology and Bioengineering
production of super-coiled plasmid DNA produced by the
non modified E. coli BL21 was 1.36 mg/g (wet weight) and
from the BL21 endA?0.87 mg/g (wet weight) compared
with about 10 mg/g (wet weight) from both BL21 recA?and
E. coli BL21 endA?/recA?.When growing on glycerol as
carbon source, the specific production of E. coli BL21 recA?
and E. coli BL21 endA?/recA?was 30% higher than in E. coli
DH5a, 10 mg/g cells (wet weight) compared with 7 mg/g
(wet weight). The volumetric production was 1.9 g/L in the
E. coli BL21 endA?/recA?culture compared with 1 g/L by
the E. coli DH5a culture. The production by the mutant
E. coli BL21 strain was not affected when glycerol was
replaced by glucose; the production was 9 mg/g cells (wet
weight) and 1.07 g/L. However, when utilizing glucose as
carbon source, E. coli DH5a production and growth were
weight) and the production in the media wasonly 100 mg/L.
The plasmid produced by the mutant E. coli BL21 was found
to be identical to the plasmid produced by E. coli DH5a as
determined by its physical properties and by its capability to
express gp120 from 293T cells. Replacing the production
strain allows us to achieve close to 90% increase in
volumetric yield. This approach together with the ability to
Funding was provided by the intramural program at the NIDDK,
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