Recombinant protein expression in escherichia coli bl21 in deuterated medium

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Technical field:The invention relates to a fed-batch method of cultivation of E. coli strain BL21(DE3) in deuterated growth media and expression of recombinant biomolecules by using a mixture of acetate and succinate as carbon sources and controlling the concentration of acetate and succinate for providing suitable conditions for such growth.

Background art:The gram-negative bacterium Escherichia coli (E. coli) is the most commonly used organism for heterologous protein production. E. coli systems are also most commonly used for industrial and pharmaceutical protein production. E. coli strain BL21 and its derivatives are the most, frequently used (Terpe, 2006). E. coli BL21(DE3)is a host bearing the T7 RNA polymerase gene (λDE3 lysogen) for expression of target proteins typically under control of the IPTG-inducible /αcUV5 promoter. The T7 polymerase system enables tight control of expression. The other big advantage of the strain BL21 is being deficient in both Ion and ompT proteases. Several expression systems have been developed for the expression of different types of proteins in E. coli BL21 .

For structural studies the proteins, peptides or other recombinant DNA products are isotopically labeled in vivo by growing the cells in media enriched with the corresponding isotope: mainly 13C, 15N and 2H (deuterium). Isotopic labeling with 2H (deuteration) of the biomolecules is advantageous while applying various methods of nuclear magnetic resonance (NMR) (Gardner and Kay, 1998), neutron scattering (Stuhrmann and Nierhaus, 1996) or mass spectrometry (MS) (Schuker and Bartch, 1994). The enrichment of the cells and recombinant products with deuterium is achieved by feeding the cells with perdeuterated (i.e., fully deuterated) carbon sources in the environment of heavy water (2H2O). In order to obtain fully deuterated biomass both the heavy water and all the substrates used should also be fully deuterated (Lederer et al, 1986). Labeling of cells and the recombinant products with deuterium is a very complicated task because deuterated environment has negative effect on the growth and severely distorts the physiology (Katz and Crespi, 1966; Gardner and Kay, 1998), and the deuterated substrates and heavy water are very expensive. This in turn necessitates to select the deuterated substrates according to their price and to apply high cell density fermentation methods as this helps to economize the use of very expensive heavy water. Even in cases of production of non-labeled heterologous proteins or in labeling with isotopes other than deuterium it is usually preferred to achieve high cell density for which different methods of fed-batch cultivation have been developed (Lee, 1996). Up to now there are a couple of publications about cultivating E. coli in heavy water in fed-batch culture (Vanatalu et al, 1993 , Jϋnemann et al, 1996) using the strain E. coli MRE600 and applying the feed- forward type of fed-batch cultivation (Paalme et al, 1990) where the overfeeding is avoided by periodically analyzing the growth medium with high performance liquid chromatograpy (HPLC) which is a laborious task as the cultivations usually last several days. The authors of this invention have demonstrated the possibility to apply a . special growth control algorithm which enabled to achieve maximum growth rates in continuous cultivation (Vanatalu, 1997) including the growth of E. coli MRE600 on succinic acid in a fully deuterated medium .

On the other hand the perdeuterated carbon sources are also very expensive. Therefore, there is a need for a suitable composition of carbon sources that are not prohibitively expensive and still enable satisfactory growth.

Strains of E. coli have been grown in heavy water with different deuterated carbon sources like algal hydrolysate, glucose , succinate, acetate (Mann and Moses, 1971) or mixture of succinate and acetate .

Strain E. coli BL21 has been cultivated in highly deuterated media either on commercial deuterated hydrolyzates (Fiaux et al, 2004), or H-acetate. Of these only 2H-acetate can be taken as cheap carbon source. In all cases the authors denote the poor growth while acetate being the carbon source.

In a recent publication (Paliy and Gunasekera, 2006) the authors conclude that the growth of E. coli BL21 is very poor on the gluconeogenic substrates acetate and succinate. Thus, there is a need for a new method to gain reasonable growth rate of E. coli BL21 in highly deuterated growth media on reasonably priced deuterated substrates enabling to achieve high biomass densities and recombinant protein expression.

Disclosure of the invention

One aspect of the invention is a method of cultivation of E. coli BL21 in a fermentation medium with deuterated media in fed-batch and expression of recombinant DNA products, comprising the steps of feeding deuterated acetate and deuterated succinate into the fermentation medium; and controlling concentrations of said acetate and said succinate in the fermentation medium below the predetermined critical levels.

Another aspect of the invention is a method of cultivation of E. coli BL21 in a fermentation medium with deuterated media in fed-batch and expression of recombinant DNA products, comprising the stages of introducing a culture of said E. coli BL21 into said fermentation medium, containing a predetermined low amount of acetate and succinate; feeding a solution of deuterated acetic acid into said fermentation medium in pH-stat regime until predetermined pH level is reached; monitoring the growth of the culture by measuring the optical density of said culture; changing the feeding algorithm from pH-stat regime to adaptastat regime when the optical density reaches a predetermined level, said adaptastat regime comprising the steps of pumping of said solution into said fermentation medium for a predetermined pumping time at a predetermined first pumping speed, corresponding to a low growth rate of said culture; halting said pumping and monitoring the change of the pθ2 of said culture until sharp rise of the pθ2 occurs; resuming pumping of said solution into said fermentation medium at a modified pumping speed, said modified pumping speed being increased by a predetermined first factor compared to said first pumping speed if the sharp rise of the pθ2 occurred within a predetermined time limit and said modified pumping speed being decreased by a predetermined second factor compared to said first pumping speed if the sharp rise of the pθ2 occurred after said predetermined time limit; and repeating the steps according to adaptastat regime until the end of the fermentation process.