Download A new approach to increase yields and improve

A new approach to increase yields and improve functionality of recombinant proteins
Voted “most innovative product” at Advances
in Recombinant Protein Technology (ELRIG),
Nov., 2013
Antti Vasala, BioSilta Oy, Oulu, Finland
2000
Introduction and background
Results: increased protein yields and improved
functionality
1500
The ability to express and extract fully functional proteins in sufficient quantities from bacterial cultures is a prerequisite for many projects in which
recombinant proteins are required for structural studies, functional characterization, as assay components or for other applications. However, low
yield, poor solubility and lack of functionality are often associated with, what should be, a routine procedure.
Conventional growth media
Conventional protein production in bacterial cultures
Most frequently, bacteria are grown in shake flasks or plates using commercially-available
or ‘in-house’ adaptations of LB, TB or M9. As illustrated in Figure 1, when supplied with
a carbon source such as glucose, bacteria grow and divide rapidly at their maximum
growth rate. However, as uncontrolled growth and fast aerobic metabolism deplete oxygen and glucose levels and alter the surrounding pH, bacteria soon switch over to anaerobic metabolism.
Uncontrolled nutrient supply, accumulation of harmful metabolites, oxygen depletion and
poor pH control limit protein yields, while the time window for expression induction can be
very short, requiring frequent O.D. measurements to determine the optimum moment.
In addition, the quality of proteins expressed and modified under sub-optimal conditions
is compromised. For example, folding and other post-translational machinery, such as
chaperones or phosphorylases, may be unable to match the fast rate of synthesis. Insufficient levels of these accessory proteins, together with oxygen depletion, increase the risk
of poor solubility or low activity of the end product.
Conventional growth media
O.D.
Rapid initial growth stops
within hours limiting maximum
cell density
Rapid initial growth stops
Rethinking the approach
O.D. to cell culture
within hours limiting maximum
cell density
Figure 2 suggests that, if bacteria are supplied with the key elements to support growth,
control pH and regulate glucose availability, cells will grow steadily to reach much higher
densities. In relation to protein expression this enables induction at aLow
high
cell density and
yield of poor
quality proteins
ensures that cells continue in linear growth for many hours after induction.leading to
significantly higher yields of protein
Oxygen depleted, pH changes anaerobic metabolism
begins
Since proteins are expressed and modified under optimal metabolic
conditions
(slower
synthesis rate, improved protein folding) these higher yields should be associated with
Uncontrolled growth, fast metabolism
improved solubility and/or functionality.
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2000
1500
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Uncontrolled growth, fast metabolism
Efficient protein
expression
Glucose fed at constant rate, pH kept in
optimal range, oxygen levels maintained
Limited time window for induction – frequent OD measurements
Proteins expressed and modified under sub-optimal metabolic
conditions(microbial folding machinery incapable of matching the
fast synthesis rate), increasing risk for inclusion bodies
EnPresso
B
EnPresso
B
0
LinB controlled conditions
LinB
Cells remain in linear growth
under
DhaA DhaA
DbjA DbjA
Cells ready to induce next morning – no OD measurement
required
Fig. 2. A theoretical growth curve assuming that bacteria are
supplied with the key elements to support growth, control
Cells
ready
to induce
next morning
– noCells
OD measurement
pH and
regulate
glucose
availability.
remain in linear
required
growth, proteins are expressed under optimal metabolic
conditions
resultingand
in modified
high yields
andoptimal
improved
functionality.
Proteins
expressed
under
metabolic
Cells remain in linear growth under controlled conditions
LB LB
EnPresso
EnPresso
B B
0
LinB LinB
Proteins expressed
and modified
under optimal
metabolic expressed in E. coli grown in an
Fig. 5a. Yields
of haloalkane
dehalogenases
conditions(slower synthesis rate, improved protein folding).
EnPresso B growth system compared to yields from the same expression system
grown in commercially-available LB media.
Results courtesy of Lukáš Chrást, Masaryk University, Czech Republic
High protein yields with improved solubility
and/or activity
0.250.25
Yield increase compared to standard LB medium
0.2 0.2
66 -fold
(mg/mL)
0.150.15
0.1(mg/mL)
20 -fold
Results courtesy of:
LB LB
Human
15-1 lipoxygenase
0.15(toxic)
T. Horn, Charité, Berlin, Germany
Fusion peptide 1
0.1
0.05
LinB
DhaA
Spur Spur
DbjA
M9
EnPresso B Defined
Nitrogen-free
1.45
32.6
2.17
5.6
DH5alpha/pETM-22
2.9
0.58
11.5
0.69
4.0
DH10B/pETM-22
2.9
0.73
12.5
0.75
4.3
puc19
Continue growth in EnPresso B
a.m.
Hours
pETM-22
2
3
4
From preculture to harvest – typical LB or TB workflow
Plasmid
production – product development and optimization
•
Multiple, time-consuming steps - often outside of normal working hours
Low yields and poor quality are typical challenges associated with the production of plasmids. Data presented in this poster shows the potential
to apply a similar approach to increase yields and improve quality as that used for production of recombinant proteins. Preliminary results obtained
Inoculate
Induce
during product developmentMonitor
andODprotocol
optimization
to produce low and highODcopy number
plasmids in two of the most commonly-used E. coli
to select
expression:
Harvest
Conventional
optimal induction time
add IPTG
media
strains are presented.
5
1.Markers
1.Markers
2.LB - DH5alpha
2.LB - DH5alpha
3.EnPresso ‘plasmid’ - DH5alpha
3.EnPresso ‘plasmid’ - DH5alpha
1
2
3
4
5
4.LB – DH10B
5.EnPresso ‘plasmid’- DH10B
Fig. 7. When compared with E. coli grown in LB media, cultures grown in a prototype EnPresso ‘plasmid growth system’ produce both high and low copy number
plasmids with a higher percentage of supercoiled (covalently closed circular) plasmid implying lower levels of nicked pDNA. The controlled growth rate within the
EnPresso culture enables cells to reach a higher density before harvesting and plasmids are produced under optimal metabolic conditions – avoiding oxygen
depletion and significant changes in pH.
Protocols for small-scale production or screening applications
6 hour cultivation
0.35 mL cultures in closed Eppendorf tubes
using a Thermomixer
0.3 mL cultures in closed Eppendorf
tubes using a Vortex Genie 2
μg DNA
ng/µL
μg DNA
ng/µL
4
DH5alpha/pET15
1.1
22
1.4
28
LB
DH10B/pUC19
2.9
58
2.9
58
M9
DH10B/pET15
1.8
36
1.4
28
EnPresso B
Defined
Nitrogen-free
Table. 4. Data in the table above shows that high and low copy number plasmids can be obtained in good yields from EnPresso ‘plasmid growth system’ cultures
using controlled shaking conditions to provide adequate aeration in the closed Eppendorf tubes. Work is therefore continuing to develop reliable protocols suitable
for plasmid production in these smaller culture volumes and using alternative formats such as microplates or deepwell plates as typically required for high throughput
screening experiments.
10
20
30
40
50
0
Fig. 6a. Cellular growth of cultures with toxic target protein (cytokine fragment)
using different media – higher cell density with EnPresso B Defined Nitrogen-free
3
7
23
0
3
7 23
Time [h] after induction
0
3
7
23
Fig. 6b. SDS-gel: samples of soluble protein produced with LB, M9 and
EnPresso B Defined Nitrogen-free after different times of induction
Results courtesy of Professor Birthe Kragelund, Dept. of Biology, University of
Copenhagen, Denmark
Conclusions
• Utilizing enzyme-controlled glucose release to control the growth of E. coli cultures expressing recombinant proteins significantly increases protein yield and
improves parameters such as the functionality, activity and solubility of the final recombinant product. Protein yields have been increased significantly when compared to yields from cultures grown in conventional LB or M9 media.
• Grow overnight - induce next morning
Cells continue in
linear growth
• Results scalable to larger culture
volumes
72
Time [h]
Fig. 4. Pre-sterilized EnPresso growth
system tablets are dissolved into
sterile water - no media autoclaving is
required. The optimized protocol
reduces hands-on time – no O.D.
monitoring is required. In a typical
workflow, cultures are grown overnight,
induced the following morning and
harvested next day.
• pDNA per 50 mL culture
500-2000 µg
3.6
0
Yields of recombinant proteins from E. coli cultures grown in conventional growth media were compared with yields from the identical expression
system and E. coli strain grown in a commercially-available EnPresso growth system (BioSilta Oy). Yields from EnPresso B were compared against
yields from LB media. Yields from EnPresso B Defined Nitrogen-free (a chemically-defined growth system specifically designed for preparation of
15N-labelled
proteins)
were compared
From preculture
to harvest
within 2against
days yields from a variety of nitrogen-free growth media.
Established
protocols
followed
for
• Minimal hands-on
time – simplewere
steps within
the working
daygrowth, induction and harvesting from conventional growth media. Protocols supplied by the manufacturer
• Follow optimized protocols
were
followed for growth, induction and harvesting from each EnPresso growth system. A typical protocol is shown in Figure 4.
• No OD monitoring
Preculture overnight,
up to 18h
5.8
68
Recombinant proteins – from pre-culture to harvest within two days
• Frequent OD monitoring
• Narrow window of opportunity for induction
DH10B/pUC19
3.4
0
p.m.
11.6
DH5alpha/pUC 19
2
Methods
a.m.
3.34
• Up to 12-fold increase of high
copy number plasmid
5
1
p.m.
41.8
6
Fig. 3. EnPresso growth systems are supplied as pre-sterilized tablets ready to dissolve in sterile water. Each tablet contains polysaccharides, growth elements and
buffering components to support optimal growth and metabolism. Controlled release of glucose from the polysaccharides maintains cells in linear growth.
a.m.
1.8
1
N-labelled protein produced in greater quantities for NMR analysis
3
Grow overnight in
EnPresso B for up to 18h
3.6
Spur
Induction after 18 hrs
Preculture
6-8h
DH5alpha/pUC19
5.EnPresso ‘plasmid’ - DH10B
— Increased protein yield
Harvest
Fold increase
conditions(slower synthesis rate, improved protein folding).
— Higher cell density
OD
mg/L/OD
4.LB – DH10B
— Improved functionality
Induce expression:
add IPTG + Reagent A +
EnPresso booster
mg/L
Table 1. Data from independent labs producing recombinant proteins from E. coli cultures grown in EnPresso B. Note the ability to significantly increase yield of a
Purified
enzymes
Purified
enzymes
Purified enzymes
lipoxygenase – previously
expressed
in very low amounts due to the toxicity of the protein.
LB
Grow culture:
add inoculum +
Reagent A
mg/L/OD
M. Crampton, CSIR, Pretoria, South Africa
0
Poor growth, low levels of expression and insufficient labelled protein are the most frequent challenges faced when using minimal medium to
prepare proteins for structural characterization by NMR analysis. Growing E. coli in EnPresso B Defined Nitrogen-free, a chemically-defined growth
system ensures that cultures can reach significantly higher densities before induction (Figure 6a). This is a particular advantage when expressing
proteins that may be toxic to E. coli. Figure 6b shows how an EnPresso growth system yielded a cytokine fragment that had been impossible to
produce using LB or M9 growth media. Table 2 shows further examples of labelled proteins prepared for NMR analysis in greater quantities than
had been achievable with other defined growth media.
Control of pH
mg/L
• Minimum 4-fold increase of low
copy number plasmid
EnPresso B
M. Wear, Edinburgh Protein Production Facility,
UK
yields
associated
with poor
Highmicrobial
protein cultures
yields with
improved
solubility
EnPresso® growth systems offering aLow
novelprotein
approach
to control
the feeding,
and thereby growth, of
in order
to achieve
high
solubility and/or low activity
and/or activity
density cell cultures, predictable induction times and hence higher yields of functional proteins. After dissolution in sterile water and addition of the
inoculum, a glucose-releasing agent breaks down long chains of polysaccharides which were contained within each tablet, releasing glucose units
at an optimal rate to feed the microbes. Controlling glucose release (the ‘feed’) controls the rate of growth enabling microbes to remain in linear
growth. Each tablet also contains key elements to support growth and maintain pH thereby ensuring optimal metabolic conditions for protein
expression.
Elements to support growth
Prototype EnPresso ‘plasmid growth system’
LB
Technology principle
Control of glucose feed
LB media
Improved quality compared to plasmids from cultures grown in LB media
15
y
50 mL culture in 500 mL Erlenmeyer flask
Spur Spur
0.25
Protein
0.2
EnPresso B
DbjA DbjA
DbjA DbjA
Fig. 5b. Specific activities of purified haloalkane dehydrogenases expressed
in E. coli and grown in an EnPresso B growth system compared to the specific
activities using the same expression system and purification method, but grown
in commercially-available LB media. Specific activity determined with
1,2-dibromoethane (LinB, DhaA and DbjA) and 1-bromobutane (Spur).
EnPresso B
Human
cyclophilin-A
0.050.05
>15 -fold (mg/mL)
0 0
Increased yields of high and low copy number plasmids from E. coli shake flask cultures
Table. 3. When compared with E. coli cultures grown in LB media, cultures grown in a prototype EnPresso ‘plasmid’ growth system’ provide significantly higher yields
of high copy number (pUC19) and low copy number (pETM-22) plasmids. Controlled growth rate within the EnPresso culture enables cells to reach higher densities
before harvesting. There are also indications that the copy number per cell is increased.
Purified
enzymes
Purified
enzymes
Purified
enzymes
Purified
enzymes
DhaA DhaA
DhaA DhaA
Spur Spur
0.1
Controlled growth, controlled metabolism
LB
0
LinB LinB
Protein yield easily compromised by uncontrolled nutrient supply,
accumulation of harmful metabolites, oxygen depletion and poor
pH control
LB
500500
Low protein yields associated with poor
solubility and/or low activity
20000.25
0.2
1500
0.15
1000
0.1
5000.05
Glucose fed at constant rate, pH kept in
optimal range, oxygen levels maintained
Controlled growth, controlled metabolism
1000
1000
0
Low yields and poor quality are typical challenges associated with the production of plasmids in shake flask cultures. Work is therefore ongoing
in the BioSilta laboratories to investigate the potential of optimizing an EnPresso growth system to increase yields of high quality plasmids from
E. coli cultures. Preliminary results obtained during protocol development to produce low and high copy number plasmids in two of the most
commonly-used E. coli strains are presented below.
>3.3-fold increase in specific activity
Efficient protein
expression
Oxygen depleted, pH changes anaerobic metabolism begins
Proteins expressed and modified under sub-optimal metabolic
conditions(microbial
machinery
incapable of matching the
Growthfolding
continues
-cultures
O.D.
fast synthesis reach
rate), increasingly
increasing risk
for inclusion
bodies
higher
cell
densities
EnPresso B
500
Figure 5a shows the substantial increases in yield of several haloalkane dehalogenase variants when E. coli cultures were grown in an EnPresso B
0
EnPresso
growth
systems
growth
system. By
controlling glucose availability, the bacteria were kept in linear growth, making
LinB it possible to reach cell densities 15-30-fold higher
DhaA
DbjA in protein functionality, in this
than those achievable in LB media before induction of protein expression. Figure 5b demonstrates the improvement
Spur
case measured in terms of specific activity, since proteins are expressed under optimal metabolic conditions. During this work it was demonstrated
enzymes
that the expression profile remained constant during the 24 hour induction period (result not shown),Purified
a contributory
factor leading to the significant
Growth continues -cultures
O.D. improvement
in yield of active enzyme per culture volume. Table 1 shows further examples of substantial increases in protein yield from cultures
reach increasingly higher cell
grown
in EnPresso B.
densities
Low yield of poor
quality proteins
Limited time window for induction – frequent OD measurements
LB
Enzymes expressed in greater amounts with higher 1000
specific activity
>30-fold increase in active enzyme
Fig. 1. Typical bacterial growth curve in conventional growth
Protein yield easily compromised by uncontrolled nutrient supply,
media. Narrow
time windows
for induction
and proteins
often
accumulation
of harmful
metabolites,
oxygen depletion
and poor
EnPresso
growth systems
expressed
pH
control under sub-optimal metabolic conditions result in
low yields associated with poor solubility and/or functionality.
Preliminary results: increased yields of high quality plasmids
Yield increase compared to standard growth media (mg/mL)
Protein
Results courtesy of:
• Independent laboratories are demonstrating that, by optimizing bacterial growth conditions using an EnPresso growth system, even proteins that would
normally be toxic to the cultures can be expressed successfully.
64-fold
Protein XX
B. Davis, Vernalis, UK
• Scientists using EnPresso B Defined Nitrogen-free are easily producing sufficient quantities of 15N-labelled proteins from single cultures prior to NMR analysis.
4 to 5-fold
Three target proteins
Major pharmaceutical company, Italy
5-fold
SPSB2 as GST fusion
E. Leung, Monash Institute of Pharmaceutical
Sciences, Australia
5-fold cf. complex medium
7-fold cf. complex medium
Protein X
Protein Y
Major pharmaceutical company, UK
>3-fold cf. modified M9
>2-fold cf. modified M9
PDZ domains as fusions
A. Diehl, Leibniz Institute for Molecular
Pharmacology, Germany
Table 2. Data from independent labs producing 15N-labelled proteins for NMR analysis from E. coli cultures grown in EnPresso B Defined Nitrogen-free
• Preliminary data applying a similar approach to increase yields of high and low copy number plasmids from E. coli cultures suggest that utilization of an EnPresso growth system optimized for plasmid production could not only enable cultures to reach higher cell densities but may also increase the plasmid number per
cell. Both factors would contribute to higher yields, providing a more cost-effective, time-efficient solution for routine plasmid production.
Acknowledgments
BioSilta would like to express their appreciation to all scientists who have contributed during the development and testing of EnPresso growth systems and who have allowed their results to be presented in this poster.
EnPresso is a trademark of BioSilta Oy