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Key stress factors and parameters for batch production
optimisation of silk-elastin-like proteins in E. coli Tony Collins, João Azevedo-Silva, André da Costa, Raul Machado, Margarida Casal
Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
Conclusions
Maximum SELP production in shake flasks was obtained with terrific broth at 37ºC, pH7.0 with a liquid vol.:vessel vol. ratio of 1:10 and an agitation speed of 200 rpm.
Maximum induction is obtained at the end of the declining exponential phase with an induction period of at least 4 hours. This allows for high cell density on induction and
reduces the rate of plasmid loss after induction.
Production levels of approximately 500 mg/L were obtained after purification.
Rapid degradation of the selection agent used, plasmid instability on induction and high acetate byproduct formation are the principal factors preventing further improved
production levels with the expression system used.
We are presently investing the fed-batch approach for overcoming some of these restraints and to further increase production levels of the novel SELP.
Introduction - Silk-elastin-like proteins (SELPs) are a family of biopolymers based on the highly repetitive amino acid sequence blocks of the naturally occurring fibrous proteins silk and elastin..
- In contrast to conventional synthetic polymers, the composition, sequence and length of these biopolymers can be strictly controlled, leading to monodispersed, precisely defined
polymers which can be biosynthesised in an ecologically friendly manner, are biodegradable and biocompatible.
- SELPs combining the physicochemical and biological properties of the high tensile strength silk with highly resilient elastin allow for the fabrication of diverse materials with a high
potential for use in the pharmaceutical, regenerative medicine and materials fields, yet their development for use is restrained by their typically low production levels.
- A series of novel SELPs composed of multiple blocks of the silkworm silk consensus sequence GAGAGS in various combinations with a mutated variant (VPAVG) of the natural
mammalian elastin repetitive sequence block VPGVG have recently been prepared.
- The genes encoding the newly designed SELPs have been synthesised and the novel recombinant polymers expressed in E. coli by use of the pET25b-E. coli BL21(DE3) expression
system.
Aims - To optimise the shake flask production levels of the novel SELP (S5E9)10 by means of a comprehensive empirical approach examining all process variables (see 1 to 4 below).
- To obtain a better understanding of the factors limiting further improved recombinant protein production of shake flask cultivations with the expression system used (see 5 to 7 below).
1. Culture Medium
• Highest biomass and SELP
production were observed with rich
buffered media such as terrific broth
(TB) and super broth (SB).
• Variation of the sugar supplement in
TB, modification of its ingredient
concentrations, or use of additives
such as ammonium, various amino
acids, magnesium or NaCl did not
improve production levels further.
2. Temperature
• 37 – 42ºC allowed for highest
production levels
• Highest biomass production was
observed at 25ºC but SELP production
was low, possibly due to repression of
the T7-based expression system at this
temperature.
• Upshifts in temperature on induction
did not lead to significantly higher
production levels.
0
2
4
6
8
10
12
14
16
18
0
20
40
60
80
100
120
LB LBM
TB TBlac
TBm
od
TBaim
SB Sbm
od
SBaim
Sben
rich
SOC
NB
S
NB
Smo
d
ZYB
ZYBb
uff
M9
Ries
Max. A
60
0n
m, M
in. p
H
SELP
Pro
du
ctio
n: %
of
Max
.
Medium SELP Prod.: % of Maximum Max. A600nm Minimum pH
0
5
10
15
20
0
20
40
60
80
100
120
25 30 37 42 25-30 25-37 30-37 37-30 37-42
Maxu
mu
m O
D6
00
nm
SELP
Pro
du
ctio
n: %
of
Max
.
Temperature
PBP Prod. (% of Maximum) Maximum OD600nm
3. Available Oxygen
• Increasing aeration, or oxygen transfer efficiency, as
indicated by increasing flask vol. to liquid vol. ratio
and increasing agitation, correlated with improved
biomass and SELP levels up to a limit.
• At higher aeration rates toxic byproduct production
(e.g. acetate) due to the higher growth rates observed
probably interfere with production.
0
2
4
6
8
10
12
14
16
0
20
40
60
80
100
120
0 5 10 15 20
Maxim
um
OD
60
0n
m
SELP
Pro
du
ctio
n: %
of
Max
.
Ratio of Flask Volume to Medium Volume
Flask Volume: Medium Volume Optimisation
SELP Production: % of Maximum Max. A600nm
0
2
4
6
8
10
12
14
16
0
20
40
60
80
100
120
80 120 160 200 240
Maxim
um
OD
60
0n
m
SELP
Pro
du
ctio
n: %
of
Max
.
Agitation (rpm)
Agitation
SELP Production: % of Maximum Max. A600nm
Fig. 4: - Effect of
varying the agitation
rate on biomass and
SELP production..
Fig. 3 - Effect of varying
the flask volume to culture
medium volume ratio on
biomass and SELP
production.
4. Induction
• In contrast to most studies which induce
during the mid-log phase, we observed
maximum production with induction at the
end of the declining exponential phase.
• At least four hours induction is
recommended for maximum production.
0
2
4
6
8
10
12
14
16
0
20
40
60
80
100
120
2 4 6 8 10 14
Maxim
um
OD
60
0n
m
SELP
Pro
du
ctio
n: %
of
Max
.
Time of Induction (hrs.)
Induction Time and Induction Period
SELP Production: 2 hrs. Induction SELP Production: 4 hrs. Induction
SELP Production: 6 hrs. Induction Maximum OD600nm
0,1
1
10
0 2 4 6 8 10 12 14
OD
60
0n
m
Time (hrs.)
Growth Curve of Uninduced E.coli BL21(DE3)
OD600nm
5. Acetate Production
• Glycerol levels rapidly decrease during the process with a concomitant
accumulation of acetate to approximately 5 g/l and a decrease of pH.
• At low glycerol concentrations the cells switch to a utilisation of the accumulated
acetate with an accompanying pH increase. This accumulated acetate provides the
carbon source during recombinant protein production.
• As little as 1 g/L acetate has a bacteriostatic effect while concentrations higher
than 4 g/L have a bactericidal effect on the host under the conditions used.
0,1
1
10
100
0
1
2
3
4
5
6
7
8
9
0 2 4 6 8 10 12 14 16 18 20 22 24 26
OD
60
0n
m
Co
nce
ntr
atio
n (
g/L)
Time (hrs) Glycerol Acetate pH OD600nm
0,1
1
10
0 2 4 6 8 10 12
OD
60
0n
m
Time (hrs.)
TB 0.5 g/l HOAc 1.0 g/l HOAc 1.5 g/l HOAc 2 g/l HOAc
3 g/l HOAc 4 g/l HOAc 5 g/l HOAc 6 g/l HOAc
7. Plasmid Stability
• Induction with IPTG leads to rapid loss of
plasmid.
• Plasmid stability remains at maximum for the
duration of cultivation in the absence of
induction.
• Induction at the later stages of the growth
curve leads to slower loss of plasmid.
0
20
40
60
80
100
0 2 4 6 8 10 12 14 16 18 20
% p
lasm
id b
ear
ing
cells
Incubation Time (hrs.)
2h 4h 8h Non Induced Cultures
Fig. 6: Monitoring of glycerol (HPLC), acetate
(HPLC) and biomass levels (OD600nm) as well as
pH as a function of incubation time using the
optimised process conditions for SELP production.
The vertical line at 8 hours marks the point of
induction with 0.5mM IPTG.
Fig. 7 - Effect of acetic acid , added at 0 hours, on
growth of E. coli BL21 DE3(+)/pET25b/SELP3
under the optimised shake flask conditions of the
present study.
6. Selection Agent Concentration
• The ampicillin concentration rapidly decreases
during the first hour of cultivation and is
already depleted at a culture OD600nm of
approximately 0.2.
• β-lactamase encoded on the pET expression
vector used leads to the observed degradation of
the selection agent.
• Productions without the use of a selection agent
have allowed for similar SELP production
levels to those with ampicillin.
Fig. 1: Media Optimisation. SELP production,
expressed as a percentage of the maximum, biomass
production (OD600nm) and minimum pH measured, as
a function of the culture medium used. SELP
production levels evaluated by SDS-PAGE.
Fig. 2: Effect of temperature on biomass and SELP
production. The right hand side of the curve (25-30, 25-
37, 30-37, 37-30, 37-42) represent the temperature shift
experiments: the initial temperatures used and the
temperatures used after induction.
Fig. 5 - Effect of induction time and induction period on
biomass and SELP production.
SELP and biomass production as a function of IPTG induction time
(2-14 hrs. incubation) and induction period (2, 4 and 6 hrs.) (top).
Growth curve of uninduced E.coli BL21(DE3) for comparison of
the induction time with the stage of growth (bottom).
Fig. 8: Ampicillin concentration (measured by the Kirby Bauer
assay) and biomass levels as a function of incubation time Fig. 8: Plasmid stability, with and without induction, at various
time points during the cultivation.
Cultures induced at 2, 4 and 8 hours (as indicated by the arrow) as
well as non-induced cultures are compared.
0 20 40 60 80 100
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
0,4
0
50
100
150
200
0 20 40 60 80 100
OD
60
0n
m
Am
pic
illin
Co
nce
ntr
atio
n (
µg/
ml)
Incubation Time (mins.) Amp. Concentration OD600nm
This work was financed by the European Commission via the 7th Framework Programme Project EcoPlast (FP7-NMP-2009-SME-3, collaborative project number 246176).