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High-level fed-batch fermentative expression of an engineered Staphylococcal protein A based ligand in E. coli: purification and characterization.

Kangwa M, Yelemane V, Polat AN, Gorrepati KD, Grasselli M, Fernández-Lahore M - AMB Express (2015)

Bottom Line: Initial fermentation of E. coli BL21 DE3:pAV01 in baffled flasks showed that high cell density was achieved when using complex media, Luria-Bertani (LB) and Terrific medium broth (TB) (10 and 14 g/L wet weight, respectively), as compared to mineral media M9, modified minimal medium (MMM) and Riesenberg mineral medium (RM) (7, 8 and 7 g/L, respectively).High cell density fed batch fermentation was achieved by selecting the best media and growth conditions, by utilizing a number of fermentation parameters like media, fermentation conditions, chemical concentrations, pO2 level, stirrer speed, pH level and feed media addition.It is possible to reach cell densities higher than shake flasks and stirred tank reactors with the improved oxygen transfer rate and feed.

View Article: PubMed Central - PubMed

Affiliation: Downstream Bioprocessing Laboratory, Department of Life Sciences and Chemistry, Jacobs University, Campus Ring 1, 28759, Bremen, Germany.

ABSTRACT
The major platform for high level recombinant protein production is based on genetically modified microorganisms like Escherichia coli (E. coli) due to its short dividing time, ability to use inexpensive substrates and additionally, its genetics is comparatively simple, well characterized and can be manipulated easily. Here, we investigated the possibilities of finding the best media for high cell density fermentation, by analyzing different media samples, focusing on improving fermentation techniques and recombinant protein production. Initial fermentation of E. coli BL21 DE3:pAV01 in baffled flasks showed that high cell density was achieved when using complex media, Luria-Bertani (LB) and Terrific medium broth (TB) (10 and 14 g/L wet weight, respectively), as compared to mineral media M9, modified minimal medium (MMM) and Riesenberg mineral medium (RM) (7, 8 and 7 g/L, respectively). However, in fed-batch fermentation processes when using MMM after 25 h cultivation, it was possible to yield an optical density (OD600) of 139 corresponding to 172 g/L of wet biomass was produced in a 30 L TV Techfors-S Infors HT fermenter, with a computer controlled nutrient supply (glucose as a carbon source) delivery system, indicating nearly 1.5 times that obtained from TB. Upon purification, a total of 1.65 mg/g of protein per gram cell biomass was obtained and the purified AviPure showed affinity for immunoglobulin. High cell density fed batch fermentation was achieved by selecting the best media and growth conditions, by utilizing a number of fermentation parameters like media, fermentation conditions, chemical concentrations, pO2 level, stirrer speed, pH level and feed media addition. It is possible to reach cell densities higher than shake flasks and stirred tank reactors with the improved oxygen transfer rate and feed.

No MeSH data available.


Related in: MedlinePlus

a A chromatographic runs of AviPure purification with 5 mL Ni–NTA column, (1) load, (2) wash, (3) elution fraction. bCoomassie Blue stained 15 % SDS-PAGE gel from different sample fractions, Lane M represent pre-stained protein marker, Lane 1 induced pellets from culture, Lane 2 supernatant after sonication and centrifugation, Lane 3 flow through, Lane 4 Wash, Lane 5 eluted sample before dialysis, Lane 6 eluted sample after dialysis
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Fig3: a A chromatographic runs of AviPure purification with 5 mL Ni–NTA column, (1) load, (2) wash, (3) elution fraction. bCoomassie Blue stained 15 % SDS-PAGE gel from different sample fractions, Lane M represent pre-stained protein marker, Lane 1 induced pellets from culture, Lane 2 supernatant after sonication and centrifugation, Lane 3 flow through, Lane 4 Wash, Lane 5 eluted sample before dialysis, Lane 6 eluted sample after dialysis

Mentions: Before chromatographic separation, the sonically lysed cells were centrifuged to remove the insoluble particles and the 40 mL of supernatant was loaded on the Ni–NTA column and the performance of the complete purification process can be seen in Fig. 3a. From the figure, three distinctive stages can clearly be identified. The AviPure elution fractions were pooled, collected and dialyzed. The concentrations of purified protein samples were determined using a Nanodrop 2000c from Thermo Scientific (Wilmington, DE, USA) with the micro volume pedestal and measurement at a wave-length of 280 nm. A total of 1.65 mg/g of protein per gram cell biomass was obtained and the product had a purity of over 90 % as was further confirmed by 15 % SDS-PAGE with coomassie blue staining (Fig. 3b). The process yield of more than 90 % was achieved, though some bands (impurities) of higher molecular weight than AviPure were visible. Due to this, a second purification step using size exclusion chromatography (SEC) (data not shown) was carried out with the aim of getting rid of the impurities.Fig. 3


High-level fed-batch fermentative expression of an engineered Staphylococcal protein A based ligand in E. coli: purification and characterization.

Kangwa M, Yelemane V, Polat AN, Gorrepati KD, Grasselli M, Fernández-Lahore M - AMB Express (2015)

a A chromatographic runs of AviPure purification with 5 mL Ni–NTA column, (1) load, (2) wash, (3) elution fraction. bCoomassie Blue stained 15 % SDS-PAGE gel from different sample fractions, Lane M represent pre-stained protein marker, Lane 1 induced pellets from culture, Lane 2 supernatant after sonication and centrifugation, Lane 3 flow through, Lane 4 Wash, Lane 5 eluted sample before dialysis, Lane 6 eluted sample after dialysis
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4641145&req=5

Fig3: a A chromatographic runs of AviPure purification with 5 mL Ni–NTA column, (1) load, (2) wash, (3) elution fraction. bCoomassie Blue stained 15 % SDS-PAGE gel from different sample fractions, Lane M represent pre-stained protein marker, Lane 1 induced pellets from culture, Lane 2 supernatant after sonication and centrifugation, Lane 3 flow through, Lane 4 Wash, Lane 5 eluted sample before dialysis, Lane 6 eluted sample after dialysis
Mentions: Before chromatographic separation, the sonically lysed cells were centrifuged to remove the insoluble particles and the 40 mL of supernatant was loaded on the Ni–NTA column and the performance of the complete purification process can be seen in Fig. 3a. From the figure, three distinctive stages can clearly be identified. The AviPure elution fractions were pooled, collected and dialyzed. The concentrations of purified protein samples were determined using a Nanodrop 2000c from Thermo Scientific (Wilmington, DE, USA) with the micro volume pedestal and measurement at a wave-length of 280 nm. A total of 1.65 mg/g of protein per gram cell biomass was obtained and the product had a purity of over 90 % as was further confirmed by 15 % SDS-PAGE with coomassie blue staining (Fig. 3b). The process yield of more than 90 % was achieved, though some bands (impurities) of higher molecular weight than AviPure were visible. Due to this, a second purification step using size exclusion chromatography (SEC) (data not shown) was carried out with the aim of getting rid of the impurities.Fig. 3

Bottom Line: Initial fermentation of E. coli BL21 DE3:pAV01 in baffled flasks showed that high cell density was achieved when using complex media, Luria-Bertani (LB) and Terrific medium broth (TB) (10 and 14 g/L wet weight, respectively), as compared to mineral media M9, modified minimal medium (MMM) and Riesenberg mineral medium (RM) (7, 8 and 7 g/L, respectively).High cell density fed batch fermentation was achieved by selecting the best media and growth conditions, by utilizing a number of fermentation parameters like media, fermentation conditions, chemical concentrations, pO2 level, stirrer speed, pH level and feed media addition.It is possible to reach cell densities higher than shake flasks and stirred tank reactors with the improved oxygen transfer rate and feed.

View Article: PubMed Central - PubMed

Affiliation: Downstream Bioprocessing Laboratory, Department of Life Sciences and Chemistry, Jacobs University, Campus Ring 1, 28759, Bremen, Germany.

ABSTRACT
The major platform for high level recombinant protein production is based on genetically modified microorganisms like Escherichia coli (E. coli) due to its short dividing time, ability to use inexpensive substrates and additionally, its genetics is comparatively simple, well characterized and can be manipulated easily. Here, we investigated the possibilities of finding the best media for high cell density fermentation, by analyzing different media samples, focusing on improving fermentation techniques and recombinant protein production. Initial fermentation of E. coli BL21 DE3:pAV01 in baffled flasks showed that high cell density was achieved when using complex media, Luria-Bertani (LB) and Terrific medium broth (TB) (10 and 14 g/L wet weight, respectively), as compared to mineral media M9, modified minimal medium (MMM) and Riesenberg mineral medium (RM) (7, 8 and 7 g/L, respectively). However, in fed-batch fermentation processes when using MMM after 25 h cultivation, it was possible to yield an optical density (OD600) of 139 corresponding to 172 g/L of wet biomass was produced in a 30 L TV Techfors-S Infors HT fermenter, with a computer controlled nutrient supply (glucose as a carbon source) delivery system, indicating nearly 1.5 times that obtained from TB. Upon purification, a total of 1.65 mg/g of protein per gram cell biomass was obtained and the purified AviPure showed affinity for immunoglobulin. High cell density fed batch fermentation was achieved by selecting the best media and growth conditions, by utilizing a number of fermentation parameters like media, fermentation conditions, chemical concentrations, pO2 level, stirrer speed, pH level and feed media addition. It is possible to reach cell densities higher than shake flasks and stirred tank reactors with the improved oxygen transfer rate and feed.

No MeSH data available.


Related in: MedlinePlus