Si l d fi d tid ti di f bi t Simple defined autoinduction medium for recombinant Simple defined autoinduction medium for recombinant protein production in E coli T7 expression system protein production in E. coli T7 expression system 1 2 Zhaopeng Li 1 , Wolfgang Kessler 2 , 1 Recombinant Protein Expression, Division of Structural Biology Helmholtz Centre for Infection Research Zhaopeng Li , Wolfgang Kessler , Joop van den Heuvel 1 Ursula Rinas 13 Recombinant Protein Expression, Division of Structural Biology 2 Research Group Microbial Drugs Fermentation Facilities Helmholtz Centre for Infection Research Inhoffenstraße 7 Joop van den Heuvel 1 , Ursula Rinas 1,3 2 Research Group Microbial Drugs, Fermentation Facilities 3 I tit t fü T hi h Ch i LUH H G Inhoffenstraße 7 38124 B h i |G 3 Institut für Technische Chemie, LUH, Hannover, Germany 38124 Braunschweig | Germany zhaopeng.li@helmholtz-hzi.de INTRODUCTION Ad t f C i l INTRODUCTION Advantage of Conventional P ti d ti i tid ti i b d di i th f E li d th d i t l f l autoinduction Preculture induction Protein production using autoinduction is based on diauxic growth of E. coli under the dynamic control of lac operon autoinduction Preculture method regulatory elements in a medium with mixtures of glucose and lactose. It omits the step of adding inducer to start Preculture protein production. Here an easy to use and cost-effective defined autoinduction medium containing glucose, glycerol, Inoculation and lactose as carbon substrates and NH 4 + as sole nitrogen source without addition of amino acids and vitamins is Inoculation 4 presented. The medium was proven to work well for recombinant proteins with different properties in different Inoculation presented. The medium was proven to work well for recombinant proteins with different properties in different cultivation scales from microplate to shaker flask and bioreactor scale Induction cultivation scales from microplate to shaker flask and bioreactor scale. Harvest H t Harvest Harvest Atid ti di ti i ti U d l bilit fT DAB di Ui l li bilit fT DAB di Autoinduction medium optimization Up or down scalability of T -DAB medium Universal applicability of T -DAB medium Reporter proteins hFGF-2 GFP hPrP PPARγ Reporter proteins hFGF 2 GFP hPrP PPARγ LB T DAB LB T DAB LB T DAB LB T DAB GFP (Green Fluorescent Protein) LB T -DAB LB T -DAB LB T -DAB LB T -DAB GFP (Green Fluorescent Protein) Scale up GST GFP (GST tagged Green GST -GFP Fluorescent Protein) hFGF 2 (Human Fibroblast Medium hFGF-2 Growth Factor-2) Medium composition 23 C composition 23 o C Bulk production 30 o C Bulk production W S I W S I W S I W S I W S I W S I W S I W S I 56 20 0 60 20 5 54 19 5 78 27 4 C lti ti 37 o C Scale down 5.6 20.0 6.0 20.5 5.4 19.5 7.8 27.4 Cultivation Scale down temperature Rab1A & SidM Auto27-243 GST -GFP YopO-GST LB T -DAB LB T -DAB LB T -DAB LB T -DAB To make sure the universal applicability of the LB T DAB LB T DAB LB T DAB LB T DAB autoinduction medium, three reporter proteins (GFP, GST - GFP and hFGF-2) have been used to optimize the GFP and hFGF 2) have been used to optimize the autoinduction medium composition at three different Hi h th h t i autoinduction medium composition at three different temperatures (23 o C 30 o C and 37 o C) Carbon source High throughput screening temperatures (23 C, 30 C and 37 C). Carbon source (glucose glycerol and lactose) and nitrogen source Autoinduction medium optimization was carried out in test (glucose, glycerol and lactose) and nitrogen source (NH + ) concentrations were carefully optimized Final Autoinduction medium optimization was carried out in test tubes and shaker flasks Up and down scaling of T -DAB (NH 4 + ) concentrations were carefully optimized. Final di iti i f ll tubes and shaker flasks. Up and down scaling of T DAB medium to 15 L bioreactor and 96 deep wells plate medium composition is as follows: medium to 15 L bioreactor and 96 deep wells plate respectively were successfully carried out as follows respectively, were successfully carried out as follows. T -DAB: Terrific-Defined Autoinduction Broth: 21 2 1.0 30 2.9 g/L glucose, 11.1 g/L glycerol, 7.6 g/L lactose, 4 g/L 21.2 30 W S I W S I W S I W S I W S I W S I W S I W S I (NH 4 ) 2 HPO 4 , 13.3 g/L KH 2 PO 4 , 1.6 g/L Citric acid, 0.6 g/L 21.0 08 Final OD600 = 23.5 25 3.8 12.0 3.6 25.5 7.4 26.9 5.0 19.9 4 2 4 2 4 MgSO 4 , 0.1 g/L Fe(III) citrate, 2.1 mg/L Na 2 MoO 4 .2H 2 O, 21.0 0.8 ) 25 Legend to figure: The target proteins are described in the first row of MgSO 4 , 0.1 g/L Fe(III) citrate, 2.1 mg/L Na 2 MoO 4 .2H 2 O, 2 5 mg/L CoCl 2 6H 2 O 15 mg/L MnCl 2 4H 2 O 1 5 mg/L 20.8 %) (%) 20 Legend to figure: The target proteins are described in the first row of each SDS PAGE Growth medium and induction method are described 2.5 mg/L CoCl 2 .6H 2 O, 15 mg/L MnCl 2 .4H 2 O, 1.5 mg/L CuCl 2H O 3 mg/L H BO 33 8 mg/L 20 6 2 (% 0.6 O 2 ( 20 0 each SDS-PAGE. Growth medium and induction method are described in the second row: LB: E coli grown in LB medium induced with 0 25 CuCl 2 .2H 2 O, 3 mg/L H 3 BO 3 , 33.8 mg/L Zn(CH COOH) 2H O 14 1 mg/L Titriplex III using NaOH 20.6 O 2 CO 15 600 in the second row: LB: E.coli grown in LB medium induced with 0.25 mM IPTG at 23 o CT -DAB: E coli grown in T -DAB medium at 23 o C In Zn(CH 3 COOH) 2 .2H 2 O, 14.1 mg/L Titriplex III, using NaOH i t bi fi l Ht 68 20 4 gas 0.4 gas hFGF 2 OD6 mM IPTG at 23 C. T -DAB: E.coli grown in T -DAB medium at 23 C. In the first row below each SDS-PAGE W: Whole cellular protein S: or ammonia to bring final pH to 6.8. 20.4 Off-g Off-g hFGF-2 10 O the first row below each SDS PAGE, W: Whole cellular protein. S: Soluble part of whole cellular protein. I: Insoluble part of whole cellular 20.2 O 02 O Soluble part of whole cellular protein. I: Insoluble part of whole cellular protein. Final biomass (OD600) for all the cultivations is shown in the Influence of dissolved oxygen and temperature: 0.2 42 39 25 5 second row below the SDS-PAGE. 20.0 42 h 39 h 25 h To test the applicability of T -DAB medium to different E.coli Yield & Solubility Atid ti lti ti 19 8 0.0 0 T7 expression systems, expression of 8 recombinant Yield & Solubility of target proteins Autoinduction cultivation i T DAB di 0 10 20 30 40 50 19.8 T7 expression systems, expression of 8 recombinant proteins in three different E coli strains (E coli BL21 (DE3) of target proteins using T -DAB media 0 10 20 30 40 50 Cultivation time (hr) All li i i bi k proteins in three different E.coli strains (E.coli BL21 (DE3), E coli Rosetta 2 (DE3) and E coli BL21 CodonPlus (DE3) High Dissolved oxygen Cultivation time (hr) All cultivation parameters in bioreactor were kept constant: E.coli Rosetta 2 (DE3) and E.coli BL21 CodonPlus (DE3) RIL) were carried out in 200 ml of T DAB media in 1 8 L High Dissolved oxygen T -DAB medium, 23 o C, 350 rpm, pH=6.8, 10 L air/min. RIL) were carried out in 200 ml of T -DAB media in 1.8 L Fernbach flasks in comparison to IPTG induction using LB Fernbach flasks in comparison to IPTG induction using LB di Th i l l hd Control medium. The same expression levels were reached d i l IPTG i d d LB di Control compared to conventional IPTG induced LB medium cultivation. The final (volumetric) yields obtained in Temperature autoinduction medium were about 4 times higher than Temperature during IPTG induction in LB medium. Dissolved oxygen Low High Cultivation temperature Better cultivation performance (yield and solubility of Conclusion: T -DAB autoinduction medium was proven Better cultivation performance (yield and solubility of target proteins) are achieved at lower temperature and to work well for E. coli T7 based expression systems for target proteins) are achieved at lower temperature and ith lti ti l hi h id b tt a broad range of proteins with different features. with cultivation vessel which can provide better oxygenation. In principal, using ammonia for pH OD600 adjustment of T -DAB medium to raise the nitrogen OD600 concentration also improves the yield of protein of Autoinduction cultivation in 96 deep wells plate at 25 o C: References interest. 1400 rpm, 0.5 ml T -DAB medium per well. 8 different References Studier F W Protein production by auto induction in high recombinant proteins were successfully produced. Studier, F.W., Protein production by auto-induction in high density shaking cultures Protein Expr Purif 2005 41: p density shaking cultures. Protein Expr Purif, 2005. 41: p. 207 34 207-34 Li Z t l Si l d fi d tid ti di f hi h Conclusion: T -DAB medium was proven to work Conclusion: In conventional test tube and shaker flask, Li, Z., et al., Simple defined autoinduction medium for high efficiently in different scale ranging from microplate, test the autoinduction medium composition was successfully level recombinant protein production for T7-based E.coli tube, shaker flask and finally to bioreactor . optimized. expression system. submitted tube, shaker flask and finally to bioreactor . optimized. CONCLUSION OUTLOOK CONCLUSION OUTLOOK A tid ti di (T DAB) d l df ti d ti i th E li T7 t Th Atid ti it th d f it i th bi i d i An autoinduction medium (T -DAB) was developed for protein production using the E. coli T7 system. The • Autoinduction omits the procedures of monitoring the biomass increase during influence of nitrogen source concentration, temperature and dissolved oxygen concentration were carefully growth and adding inducer to start production and makes the whole studied. In principal, better results were obtained in systems with higher nitrogen source concentration, procedure easy, economic and reproducible. lower cultivation temperature and better oxygenation. Subsequently, 8 different proteins with a wide range • The cultivation reproducibility and stability of chemical defined medium is of molecular weights were successfully produced in 1.8 L Fernbach flasks with very high final biomass much better than complex medium containing nutrients which are not of molecular weights were successfully produced in 1.8 L Fernbach flasks with very high final biomass concentrations (OD600>20) and very good expression levels which were at the same level as after IPTG defined The medium developed in this study can be used for heavy isotope concentrations (OD600>20) and very good expression levels which were at the same level as after IPTG induction in LB medium The T -DAB medium was tested in different cultivation scale from 96 deep wells defined. The medium developed in this study can be used for heavy isotope, or selenomethionine labeling for protein structure determination through NMR induction in LB medium. The T -DAB medium was tested in different cultivation scale from 96 deep wells plate to test tube to shaker flask (100 ml to 2 L) and finally in a 15 L bioreactor Over expression of or selenomethionine labeling for protein structure determination through NMR spectroscopy or X ray crystallography plate to test tube, to shaker flask (100 ml to 2 L), and finally in a 15 L bioreactor. Over-expression of recombinant proteins and high final target protein yield (> 500 mg/L) was achieved in all cultivation scales spectroscopy or X-ray crystallography. recombinant proteins and high final target protein yield (> 500 mg/L) was achieved in all cultivation scales.
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Si l d fi d t i d ti di f bi tSimple defined autoinduction medium for recombinantSimple defined autoinduction medium for recombinant protein production in E coli T7 expression systemprotein production in E. coli T7 expression systemp ote p oduct o co e p ess o syste
1 2Zhaopeng Li1, Wolfgang Kessler2, 1 Recombinant Protein Expression, Division of Structural Biology Helmholtz Centre for Infection ResearchZhaopeng Li , Wolfgang Kessler , Joop van den Heuvel1 Ursula Rinas1 3
Recombinant Protein Expression, Division of Structural Biology 2 Research Group Microbial Drugs Fermentation Facilities
Helmholtz Centre for Infection ResearchInhoffenstraße 7Joop van den Heuvel1, Ursula Rinas1,3 2 Research Group Microbial Drugs, Fermentation Facilities
3 I tit t fü T h i h Ch i LUH H GInhoffenstraße 7 38124 B h i | G3 Institut für Technische Chemie, LUH, Hannover, Germany 38124 Braunschweig | [email protected] g @
INTRODUCTION Ad t f C i lINTRODUCTION Advantage of Conventional
P t i d ti i t i d ti i b d di i th f E li d th d i t l f l autoinduction Precultureinduction
Protein production using autoinduction is based on diauxic growth of E. coli under the dynamic control of lac operon autoinduction Preculture methodregulatory elements in a medium with mixtures of glucose and lactose. It omits the step of adding inducer to start Precultureprotein production. Here an easy to use and cost-effective defined autoinduction medium containing glucose, glycerol, Inoculationp p y g g g yand lactose as carbon substrates and NH4
+ as sole nitrogen source without addition of amino acids and vitamins isInoculation
4 gpresented. The medium was proven to work well for recombinant proteins with different properties in different Inoculationpresented. The medium was proven to work well for recombinant proteins with different properties in differentcultivation scales from microplate to shaker flask and bioreactor scale Inductioncultivation scales from microplate to shaker flask and bioreactor scale.
Harvest H tHarvest Harvest
A t i d ti di ti i ti U d l bilit f T DAB di U i l li bilit f T DAB diAutoinduction medium optimization Up or down scalability of T-DAB medium Universal applicability of T-DAB mediump p y pp y
Reporter proteins hFGF-2 GFP hPrP PPARγReporter proteins hFGF 2 GFP hPrP PPARγ
LB T DAB LB T DAB LB T DAB LB T DABGFP (Green Fluorescent Protein)
LB T-DAB LB T-DAB LB T-DAB LB T-DABGFP (Green Fluorescent Protein)
Scale up
GST GFP(GST tagged Green
GST-GFP( ggFluorescent Protein)
hFGF 2(Human Fibroblast
MediumhFGF-2 Growth Factor-2) Medium
composition23 C composition23 oC
Bulk production30 oC Bulk productionW S I W S I W S I W S I W S I W S I W S I W S I
5 6 20 0 6 0 20 5 5 4 19 5 7 8 27 4C lti ti
37 oC Scale down 5.6 20.0 6.0 20.5 5.4 19.5 7.8 27.4Cultivation Scale down
temperature Rab1A & SidM Auto27-243 GST-GFP YopO-GSTpLB T-DAB LB T-DAB LB T-DAB LB T-DAB
To make sure the universal applicability of the LB T DAB LB T DAB LB T DAB LB T DAB
pp yautoinduction medium, three reporter proteins (GFP, GST-, p p ( ,GFP and hFGF-2) have been used to optimize theGFP and hFGF 2) have been used to optimize the autoinduction medium composition at three different Hi h th h t iautoinduction medium composition at three different temperatures (23 oC 30 oC and 37 oC) Carbon source
High throughput screeningtemperatures (23 C, 30 C and 37 C). Carbon source (glucose glycerol and lactose) and nitrogen source Autoinduction medium optimization was carried out in test(glucose, glycerol and lactose) and nitrogen source (NH +) concentrations were carefully optimized Final
Autoinduction medium optimization was carried out in test tubes and shaker flasks Up and down scaling of T-DAB(NH4
+) concentrations were carefully optimized. Final di iti i f ll
tubes and shaker flasks. Up and down scaling of T DAB medium to 15 L bioreactor and 96 deep wells platemedium composition is as follows: medium to 15 L bioreactor and 96 deep wells plate respectively were successfully carried out as followsrespectively, were successfully carried out as follows.
T-DAB: Terrific-Defined Autoinduction Broth: 21 2
1.0 302.9 g/L glucose, 11.1 g/L glycerol, 7.6 g/L lactose, 4 g/L 21.2 30
W S I W S I W S I W S I W S I W S I W S I W S Ig g , g g y , g , g(NH4)2HPO4, 13.3 g/L KH2PO4, 1.6 g/L Citric acid, 0.6 g/L 21.0 0 8
Final OD600 = 23.5 25 3.8 12.0 3.6 25.5 7.4 26.9 5.0 19.9( 4)2 4, g 2 4, g , gMgSO4, 0.1 g/L Fe(III) citrate, 2.1 mg/L Na2MoO4.2H2O,
21.0 0.8
)
25
Legend to figure: The target proteins are described in the first row ofMgSO4, 0.1 g/L Fe(III) citrate, 2.1 mg/L Na2MoO4.2H2O, 2 5 mg/L CoCl2 6H2O 15 mg/L MnCl2 4H2O 1 5 mg/L 20.8
%) (%
)
20Legend to figure: The target proteins are described in the first row of each SDS PAGE Growth medium and induction method are described2.5 mg/L CoCl2.6H2O, 15 mg/L MnCl2.4H2O, 1.5 mg/L
CuCl 2H O 3 mg/L H BO 33 8 mg/L 20 62 (% 0.6
O2 ( 20
0
each SDS-PAGE. Growth medium and induction method are described in the second row: LB: E coli grown in LB medium induced with 0 25CuCl2.2H2O, 3 mg/L H3BO3, 33.8 mg/L
Zn(CH COOH) 2H O 14 1 mg/L Titriplex III using NaOH20.6 O
2
CO
15 600 in the second row: LB: E.coli grown in LB medium induced with 0.25
mM IPTG at 23 oC T-DAB: E coli grown in T-DAB medium at 23 oC InZn(CH3COOH)2.2H2O, 14.1 mg/L Titriplex III, using NaOH i t b i fi l H t 6 8 20 4ga
s
0.4gas
hFGF 2 OD
6 mM IPTG at 23 C. T-DAB: E.coli grown in T-DAB medium at 23 C. In the first row below each SDS-PAGE W: Whole cellular protein S:or ammonia to bring final pH to 6.8. 20.4
Off-
g
Off-
ghFGF-2 10 O the first row below each SDS PAGE, W: Whole cellular protein. S: Soluble part of whole cellular protein. I: Insoluble part of whole cellular
20.2O
0 2 O
Soluble part of whole cellular protein. I: Insoluble part of whole cellular protein. Final biomass (OD600) for all the cultivations is shown in the
Influence of dissolved oxygen and temperature: 0.2423925 5
p ( )second row below the SDS-PAGE.
20.042 h
39 h25 h
To test the applicability of T-DAB medium to different E.coliYield & Solubility A t i d ti lti ti 19 80.0 0 pp y
T7 expression systems, expression of 8 recombinantYield & Solubilityof target proteins
Autoinduction cultivation i T DAB di 0 10 20 30 40 50
19.8T7 expression systems, expression of 8 recombinant proteins in three different E coli strains (E coli BL21 (DE3)
of target proteins using T-DAB media 0 10 20 30 40 50Cultivation time (hr)
All l i i i bi kproteins in three different E.coli strains (E.coli BL21 (DE3), E coli Rosetta 2 (DE3) and E coli BL21 CodonPlus (DE3)High Dissolved oxygen
Cultivation time (hr)
All cultivation parameters in bioreactor were kept constant: E.coli Rosetta 2 (DE3) and E.coli BL21 CodonPlus (DE3) RIL) were carried out in 200 ml of T DAB media in 1 8 L
High Dissolved oxygen
T-DAB medium, 23 oC, 350 rpm, pH=6.8, 10 L air/min. RIL) were carried out in 200 ml of T-DAB media in 1.8 L Fernbach flasks in comparison to IPTG induction using LBFernbach flasks in comparison to IPTG induction using LB
di Th i l l h dControl medium. The same expression levels were reached d i l IPTG i d d LB di
Control
compared to conventional IPTG induced LB medium cultivation. The final (volumetric) yields obtained in Temperature autoinduction medium were about 4 times higher than Temperature gduring IPTG induction in LB medium.g
Dissolved oxygenLow HighCultivation temperature
g
Better cultivation performance (yield and solubility ofConclusion: T-DAB autoinduction medium was proven
Better cultivation performance (yield and solubility of target proteins) are achieved at lower temperature and
to work well for E. coli T7 based expression systems for target proteins) are achieved at lower temperature and
ith lti ti l hi h id b tt
ya broad range of proteins with different features.
with cultivation vessel which can provide better g p
oxygenation. In principal, using ammonia for pH OD600adjustment of T-DAB medium to raise the nitrogen OD600
concentration also improves the yield of protein of Autoinduction cultivation in 96 deep wells plate at 25 oC: Referencesp y pinterest. 1400 rpm, 0.5 ml T-DAB medium per well. 8 different
ReferencesStudier F W Protein production by auto induction in highp p
recombinant proteins were successfully produced.Studier, F.W., Protein production by auto-induction in high density shaking cultures Protein Expr Purif 2005 41: pp y p density shaking cultures. Protein Expr Purif, 2005. 41: p. 207 34207-34Li Z t l Si l d fi d t i d ti di f hi hConclusion: T-DAB medium was proven to work Conclusion: In conventional test tube and shaker flask, Li, Z., et al., Simple defined autoinduction medium for high p
efficiently in different scale ranging from microplate, test ,
the autoinduction medium composition was successfully level recombinant protein production for T7-based E.coli y g g p ,tube, shaker flask and finally to bioreactor.
p yoptimized. expression system. submittedtube, shaker flask and finally to bioreactor.optimized. p y
CONCLUSION OUTLOOKCONCLUSION OUTLOOK
A t i d ti di (T DAB) d l d f t i d ti i th E li T7 t Th A t i d ti it th d f it i th bi i d iAn autoinduction medium (T-DAB) was developed for protein production using the E. coli T7 system. The • Autoinduction omits the procedures of monitoring the biomass increase during influence of nitrogen source concentration, temperature and dissolved oxygen concentration were carefully growth and adding inducer to start production and makes the whole studied. In principal, better results were obtained in systems with higher nitrogen source concentration, procedure easy, economic and reproducible.y g glower cultivation temperature and better oxygenation. Subsequently, 8 different proteins with a wide range • The cultivation reproducibility and stability of chemical defined medium is p yg q y, p gof molecular weights were successfully produced in 1.8 L Fernbach flasks with very high final biomass
p y ymuch better than complex medium containing nutrients which are not of molecular weights were successfully produced in 1.8 L Fernbach flasks with very high final biomass
concentrations (OD600>20) and very good expression levels which were at the same level as after IPTGuc bette t a co p e ed u co ta g ut e ts c a e ot
defined The medium developed in this study can be used for heavy isotopeconcentrations (OD600>20) and very good expression levels which were at the same level as after IPTG induction in LB medium The T-DAB medium was tested in different cultivation scale from 96 deep wells
defined. The medium developed in this study can be used for heavy isotope, or selenomethionine labeling for protein structure determination through NMRinduction in LB medium. The T-DAB medium was tested in different cultivation scale from 96 deep wells
plate to test tube to shaker flask (100 ml to 2 L) and finally in a 15 L bioreactor Over expression ofor selenomethionine labeling for protein structure determination through NMR spectroscopy or X ray crystallographyplate to test tube, to shaker flask (100 ml to 2 L), and finally in a 15 L bioreactor. Over-expression of
recombinant proteins and high final target protein yield (> 500 mg/L) was achieved in all cultivation scalesspectroscopy or X-ray crystallography.
recombinant proteins and high final target protein yield (> 500 mg/L) was achieved in all cultivation scales.
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