E. coli Host Case Study: ScarabXpress-1(T7lac) vs BL21
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E. coli Host Case Study:
ScarabXpress®-1(T7lac) vs BL21
SCARABXPRESS®-1(T7LAC) YIELDS 12X MORE PROTEIN THAN BL21
Scarab Genomics has generated a series of multiple deletion strains (MDS) of E. coli that lack numerous non-
essential genes, mobile DNA elements (insertion sequences (IS) and prophages), and potential virulence
factors. This Clean Genome® E. coli MDS platform provides bacterial strains with ideal characteristics for
regulated biopharmaceutical applications:
Scarab Genomics engineered its Clean Genome® E. coli hosts for robust growth in minimal salts
media, allowing production under strictly defined conditions.
Plasmid and genomic stability are enhanced because transposable insertion sequences have been
eliminated.
Fermentations are more stable because cryptic prophage were deleted, eliminating cell lysis.
Genes for toxins, virulence factors, flagella and fimbriae have also been removed to improve
product purity and safety.
Cells can continue to grow during protein expression because over 700 non-essential genes are
eliminated and no longer compete for cellular resources. Increasing metabolic efficiency
Increased percentage yield (up to 40% TCP) drives down post fermentation processing costs.
These strains can be further engineered for use in customer-specific protein expression systems. In this
paper, we describe the process development procedure used to improve the production of an economically
important protein from 1.2 g/L to >14 g/L using a Clean Genome® E. coli expression strain.
ScarabXpress®-1 Bacterial Strain: An Improved Protein Expression Host.
A well-characterized multiple deletion strain lacking 15% of the native genome (Figure 1) was used to create a
pET expression host by genomic insertion of the gene for T7 RNA polymerase under the control of a modified
lac promoter and operator. The resulting ScarabXpress®-1 strain has more tightly regulated protein expression
than the commonly used BL21(DE3), providing the ability to reproducibly and precisely control induction. The
tightly regulated induction of target gene expression in ScarabXpress®-1 permits continued cell growth after
protein induction because the expression has more subtle effects on cellular physiology and is thus less likely
to induce cellular stress responses. Since the T7 polymerase is encoded in the genome, the strain remains
prophage-free. These advantages, in combination with a Clean Genome® E. coli strain background free of IS
elements and prophage, make an ideal protein expression system.
www.scarabgenomics.com | 888.513.7075 | info@scarabgenomics.com
Figure 1
Genome maps of
BL21(DE3) and
ScarabXpress
Color key:
deletions (black),
prophage (green)
and IS elements
(red), RNA operons
(purple) and genes
(blue and yellow)
CASE STUDY
Increased specific yield of a test protein using ScarabXpress®-1 as the host strain.
A pharmaceutical company was in the process of scaling up production of a protein biologic (TEST-PRO) for
evaluation in a clinical trial. TEST-PRO is an 83-kDa cytoplasmic protein of human origin. The company
utilized the pET24 expression vector and E. coli BL21(DE3) as the host strain. Fermentation of
BL21(DE3)/pET24-TEST-PRO was considered inadequate to meet the needs of the clinical trial, as the
required amount of pharmaceutical product exceeded production capabilities.
In an effort to increase expression and decrease cost, the company collaborated with Scarab Genomics to
evaluate the Clean Genome® E. coli technology for protein production. The pET24-TEST-PRO plasmid in
ScarabXpress®-1 produced at least twice as much protein compared to BL21(DE3) (see Table 1). Further
optimization with the ScarabXpress®-1 construct, resulting in greater than ten-fold increase in protein
production, was achieved by using a more robust expression vector and by optimizing the medium, IPTG
concentration, and time of induction.
Evaluation of Vector/Host Combination
Some pET vectors have additional control elements regulating target protein expression. Plasmids with fewer
regulatory elements generally give highest induced expression, but are encumbered by unregulated levels of
basal (background) expression. Conversely, plasmids containing more elaborate regulatory regions exhibit low
basal expression, but are more difficult to induce fully. We evaluated induced and uninduced levels of TEST-
PRO expression using two hosts and two plasmids. The coding region for TEST-PRO was inserted in the less
tightly regulated pET9 vector, as well as the highly controlled pET24. Each plasmid was transformed into
ScarabXpress®-1 as well as BL21(DE3). Upon induction, higher levels of TEST-PRO were obtained with pET9
compared with pET24 (summarized in Table 1), so pET9 was chosen for further optimization.
2BL21(DE3) (M) ScarabXpress®
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
150
100
75
50
37
25
20
15
10
IPTG (μM): 0 10 25 50 100 200 400 1000 0 10 25 50 100 200 400 1000
3
OD600
@Harvest 0
Figure 2. Inducer titration of pET9-TEST-PRO expression in BL21(DE3) and ScarabXpress®.
Shake flask cultures were grown in 50 ml Korz minimal medium plus 0.2% glucose at 37°C to an OD600 of 0.01 then induced
overnight with the indicated concentrations of IPTG. Cell pellets from each sample were obtained by centrifugation and soluble
proteins were resuspended in BugBuster® protein extraction reagent for SDS-PAGE analysis, loading the same OD600 equivalent
in each lane.. (M), molecular mass marker (sizes in kd at left edge of gel); arrowheads indicate TEST-PRO protein; chart at
bottom indicates cell density (OD600) at harvest.
An examination of the basal levels of expression in the two strains revealed that in ScarabXpress®-1, there
was no detectable TEST-PRO expressed from the pET9 construct (Figure 2, lane 10). In contrast, there was a
significant level of TEST-PRO from BL21(DE3) in the absence of IPTG (Figure 2, lane 1). Furthermore, a
comparison of the induction profile of the two strains, with varying concentrations of IPTG, indicated that higher
levels of TEST-PRO were obtained with ScarabXpress®-1. Thus, ScarabXpress®-1 appears to be the ideal
host strain since it combines the advantage of tight regulatory control with high inducible levels of target protein
expression.
Growth and induction conditions
Fermentations using fully defined media are generally preferred by the FDA for biopharmaceutical
manufacturing. However, the carbon source in the medium strongly influences regulatory control and target
protein production, and use of minimal media with traditional E. coli hosts can be problematic.
ScarabExpress®-1 strains, in contrast, grow well and maintain tight regulatory control in a variety of defined
minimal media, including Korz minimal medium with glucose or glycerol. For this development project, Korz
with glucose was used in shake flask optimizations and for scale up in fed batch fermenters.
3The question of how much inducer is needed for optimal induction was addressed by titration of shake flask
cultures with IPTG. The goal was to determine the amount of IPTG that would provide a high level of
expression without compromising growth. BL21(DE3)/pET9-TEST-PRO and ScarabXpress®-1 /pET9-TEST-
PRO were grown in Korz minimal medium with 0.2% glucose. Cultures were induced early in growth (OD600 of
0.01) with IPTG ranging from 0 to 1 mM (Figure 2). ScarabXpress®-1 cells showed high protein expression at
inducer concentration as low as 10 μM, with good protein expression over the entire IPTG concentration range.
The level of expression in BL21(DE3) did not peak until 25 μM IPTG was used for induction, with expression
declining then stabilizing to a lower level with higher concentrations of IPTG.
Although the induction profile of TEST-PRO in ScarabXpress®-1 appeared to be more stable and yielded
significantly more protein than that of BL21(DE3) in response to increasing levels of IPTG, an inhibitory effect
of IPTG on overnight growth was observed that was not observed with BL21(DE3) (Figure 2). To examine this
more closely, ScarabXpress®-1 /pET9-TEST-PRO was induced with different concentrations of IPTG and
OD600 was monitored at 1-minute intervals throughout the entire growth of the culture (Figure 3). Growth of the
culture was largely uninhibited by concentrations of IPTG up to 10 μM whereas at 15, 20, or 25 μM IPTG
growth was significantly inhibited. In order to assess the amount of TEST-PRO expression in each culture,
samples were removed at regular intervals throughout growth and analyzed by SDS-PAGE. As can be seen in
Fig. 3b, IPTG concentrations of 10 μM and greater had the steadiest induction profile, with a gradual
accumulation of TEST-PRO over time. In contrast, lower concentrations of IPTG did not yield appreciable
amounts of TEST-PRO until late in growth. The results of the titration indicate that induction with 10 μM IPTG
is optimal for high-level protein synthesis with no effect on growth.
Figure 3. Growth and Induction of ScarabXpress/pET9- OD 600
TEST-PRO 1
Shake flask cultures were grown in 50 ml Korz minimal
medium plus 0.2% glucose at 37°C, with monitoring of OD600
at 1-minute intervals. The indicated concentrations of IPTG 0.1 0 M IPTG
were added to the cultures at an OD600 of 0.01 1
2.5
Fig. 3a (right): Growth at indicated IPTG concentrations 5
10
Fig. 3b (below): Induction profile. Samples were collected 0.01
15
for SDS-PAGE analysis at the times indicated. Cell pellets 20
were obtained by centrifugation, and soluble proteins were 25
resuspended in BugBuster® protein extraction reagent. 0.001
0 6 12 18 Hours
Hrs 6 7 8 9 o/n 6 7 8 9 o/n 6 7 8 9 o/n 6 7 8 9 o/n 5 6 7 8 o/n 6 7 8 o/n 7 8 o/n 7 8 9 o/n
IPTG 0 μM 1 μM 2.5 μM 5 μM 10 μM 15 μM 20 μM 25 μM
4ScarabXpress® TEST-PRO Fed-Batch fermentation
The results of the shake flask experiments were applied to small scale fed-batch fermentation (1.5 L initial
volume) of ScarabXpress®-1 /pET9-TEST-PRO in Korz minimal medium with glucose as the carbon source.
Induction was initiated early in the batch phase at an OD600 of 2-3 by the addition of 10 μM IPTG. Upon
exhaustion of the carbon source, an exponential feed was initiated at a specific growth rate of 0.25 h-1. The
feed medium contained glucose and 10 μM IPTG, and the rate of feed was controlled by monitoring the
concentrations of dissolved oxygen and glucose in the culture. Samples were removed at periodic intervals
and analyzed by SDS-PAGE.
Figure 4 shows the growth curves for three different fermentations. All fermentations had a high yield of cell
mass with final OD600 ~160 and ~300 g/L of wet weight. TEST-PRO production was evident approximately 9
hours after induction and >95% of the protein remained associated with the soluble fraction (Figure 4, inset).
Moreover, TEST-PRO levels remained steady as a percent of total mass for the remainder of the run.
100
Ferm 112
Figure 4. Fed-batch Ferm 108
fermentation of ScarabXpress® 10 Ferm 125 Soluble Insoluble
/pET9-TEST-PRO OD600
Growth curves for three 1
fermentations. Samples were
collected at indicated time points
for analysis by SDS-PAGE. 0.1
+ Inducer at
Soluble and insoluble fractions OD600 = 2-3
were fractionated by 0.01
centrifugation. Inset shows SDS-
PAGE data for Fermentation 108.
0.001
0 6 12 18 24 30 36 hr
Western blot analysis of the harvested material showed no degradation of TEST-PRO (data not shown),
indicating that the protein is stable in the Clean Genome® E. coli background. In addition, the high cell
densities recorded and large fermentation pellets obtained indicate that cell lysis did not occur in the
ScarabXpress®-1 fermentations. This is in contrast to fed-batch fermentation of BL21(DE3)/pET24-TEST-PRO,
where up to 50% of the protein was observed in the culture supernatant with associated cell lysis (data not
shown). This indicates that BL21(DE3) strain background is susceptible to stress-induced prophage lytic
enzymes whereas ScarabXpress®-1 is not.
Densitometric scans of SDS-polyacrylamide gels were performed to determine the percentage of total cellular
protein (TCP) represented by TEST-PRO, and the specific yield of TEST-PRO protein was calculated for three
independent runs of three different host strain / pET vector combinations (Table 1). For ScarabXpress®-1 with
the pET9-TEST-PRO plasmid, induced with 10M IPTG, the test protein from 25% to 41% of TCP, with an
average specific protein yield ranging from 13 to 16 g/L culture (Table 1).
5Table 1. Test protein yields from Fed-batch fermentations in Korz minimal medium with
glucose
Host ScarabXpress®-1 (T7lac) BL21(DE3)
Vector pET9-TEST-PRO pET24-TEST-PRO pET24-TEST-PRO
IPTG Conc. 10M 1 mM 1 mM
Fermentation # 108 112 125 98 100 101 97 99 102
Induction OD600 2.94 4.3 3.66 10.7 9.6 10.2 9.4 12.8 11.7
Harvest OD600 238 244 214 211 178 192 111 142 162
% of total cell protein 25-31 26-29 33-41 13-15 13-15 13-14 9-9 7-7 5-5
Calculated Yield, g/L 13-16 13-14 14-16 4-5 4-5 6-6 1-1 1-2 1-1
Average Yield, g/L 14.6 5.1 1.19
By comparison, fed-batch fermentation of the same ScarabXpress®-1 host with the pET24-TEST-PRO
plasmid, with a later induction (OD600 ~10) and a higher amount of IPTG (1mM) required, averaged yields of
only 5.1 g/LTEST-PRO. The lowest performing strain/vector combination was the commonly used BL21(DE3)
host with the pET24-TEST-PRO plasmid, which had an average yield of 1.2 g/L, approximately 12-fold less
than that obtained with ScarabXpress®-1 /pET9-TEST-PRO. The results indicate that the preferred fed-batch
process for TEST-PRO production should include early induction with an optimal (i.e. lower) concentration of
inducer in a Clean Genome® E. coli host.
Why is expression increased in ScarabXpress®-1?
Four key attributes of the ScarabXpress®-1 system contribute to its superior performance in biomanufacturing:
Less energy wasted—As a Clean Genome® E. coli strain, ScarabXpress®-1 has been stably deleted for
15% of the wild type genome, consequently there are >700 fewer open reading frames to compete for
resources during protein production.
Genetic stability—Both the bacterial genome and any introduced plasmid are more genetically stable in
these cells because all mobile DNA—insertion sequences, prophages and transposons—were deleted.
Phage removal—With no prophage in the bacterial chromosome, lysis from prophage induction cannot
occur during fermentation.
Tight genetic regulation—To control recombinant protein expression, the gene for T7 RNA polymerase in
these cells is under the control of a modified lac promoter/operator system. The important features of the
ScarabXpress®-1 promoter/operator controlling T7 RNA polymerase expression pertain to the strength of
the promoter, enhanced sensitivity, and degree of repression conferred by LacI repressor protein.
The superior regulation deserves discussion, as its benefits may in some respects be counterintuitive.
ScarabXpress®-1 utilizes the wild-type lac promoter, which is of lower strength relative to the lacUV5 variant
present in popular biomanufacturing host BL21(DE3). Moreover, the wild-type lac promoter is subject to
regulation by the catabolite activator protein, CAP, whereas BL21(DE3) lacUV5 activity is largely CAP-
6independent due to a change in the CAP binding site. The wild-type promoter is thus more sensitive to the
effects of catabolite repression, and promoter activation occurs gradually post-induction. This is in contrast to
the rather abrupt expression of lacUV5 upon induction that is a reflection of its strength and CAP-
independence. One might therefore have predicted superior performance from lacUV5-regulated production of
protein or DNA products, whereas the ScarabXpress®-1 system proved dramatically more efficient.
The ScarabXpress®-1 system is more sensitive to repression; the promoter is more tightly regulated by LacI
due to an alteration in the O1 binding site (Super lacO). The net result is a lowering of the background levels of
expression relative to BL21(DE3). As a consequence of this tighter regulation, the ScarabXpress®-1 host
typically works optimally with expression vectors that do NOT supply extra lac repressor from a plasmid
encoded copy of the lacI gene. In multiple cases, significantly higher expression yields of a target protein have
been observed by using a vintage pET vector – i.e. one that carries neither the lacI gene nor a lac operator on
its backbone. lacI-based pET plasmids were created to address the inherent leakiness of BL21(DE3). The
additional lac repressor generated from this type of vector prevents full induction of the ScarabXpress®-1 host
and may only yield optimal results when attempting to express a particularly toxic target protein. Therefore
users of the ScarabXpress®-1 platform will gain the full benefits only when the vector carrying the gene to be
expressed also carry the appropriate regulatory machinery.
Potential for More Standardization of Biomanufacturing Processes
Standardization of protocols for large-scale production of protein-based biopharmaceuticals has proven elusive
due to factors such as variability in the genetic makeup of production host strains, methods of expression,
control of induction, protein solubility, toxicity, and proprietary restrictions. With traditional biomanufacturing
systems, therefore, a new process must be developed and optimized for each new biopharmaceutical product,
a costly and time consuming process. While it may be difficult to envision a system that completely bypasses
the need for optimization, the development of the ScarabXpress®-1 platform shows promise toward
standardizing the process development cycle. Tight regulatory control of target gene expression greatly
facilitates optimization of manufacturing protocols at the flask culture level, and these optimized conditions
transfer largely intact upon scale up to fed-batch fermentation. Thus the value of the ScarabXpress®-1 platform
is not merely the superior yields of protein and DNA products, but also the savings in development of robust,
reliable and consistent manufacturing processes.
CONCLUSION
The ScarabXpress®-1 Clean Genome® E. coli platform delivers higher yields of biomanufactured products and
more consistent manufacturing processes than any other E. coli host system.
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