A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures

Abstract

Background: Cultivations for recombinant protein production in shake flasks should provide high cell densities, high protein productivity per cell and good protein quality. The methods described in laboratory handbooks often fail to reach these goals due to oxygen depletion, lack of pH control and the necessity to use low induction cell densities. In this article we describe the impact of a novel enzymatically controlled fed-batch cultivation technology on recombinant protein production in Escherichia coli in simple shaken cultures.

Results: The enzymatic glucose release system together with a well-balanced combination of mineral salts and complex medium additives provided high cell densities, high protein yields and a considerably improved proportion of soluble proteins in harvested cells. The cultivation method consists of three steps: 1) controlled growth by glucose-limited fed-batch to OD600 approximately 10, 2) addition of growth boosters together with an inducer providing efficient protein synthesis within a 3 to 6 hours period, and 3) a slow growth period (16 to 21 hours) during which the recombinant protein is slowly synthesized and folded. Cell densities corresponding to 10 to 15 g l(-1) cell dry weight could be achieved with the developed technique. In comparison to standard cultures in LB, Terrific Broth and mineral salt medium, we typically achieved over 10-fold higher volumetric yields of soluble recombinant proteins.

Conclusions: We have demonstrated that by applying the novel EnBase Flo cultivation system in shaken cultures high cell densities can be obtained without impairing the productivity per cell. Especially the yield of soluble (correctly folded) proteins was significantly improved in comparison to commonly used LB, Terrific Broth or mineral salt media. This improvement is thought to result from a well controlled physiological state during the whole process. The higher volumetric yields enable the use of lower culture volumes and can thus significantly reduce the amount of time and effort needed for downstream processing or process optimization. We claim that the new cultivation system is widely applicable and, as it is very simple to apply, could widely replace standard shake flask approaches.

Figure 1

Characteristics of recombinant E. coli strain RB791 [pQE30:Adh] cultivations in different culture media. EB = EnBase Flo medium, MSM = mineral salt medium, LB = Luria-Bertani medium, TB = Terrific Broth medium. A: OD₆₀₀ in 50 ml cultures in 500 ml shake flasks; B) pH in 50 ml cultures in 500 ml shake flasks; C) OD₆₀₀ in 3 ml cultures in 24-deep well plates. The time point of induction (0.4 mM IPTG) is indicated with a dashed vertical line. EnBase Flo cultures were also supplemented with "medium booster" (complex additives) at the same time with induction. EnBase Flo cultures were grown for 19 h before induction, whereas cultures in MSM, LB and TB media were grown for 2 h before induction. All cultures were run until 24 h after induction.

Figure 2

Recombinant protein production in 24 DWPs using different cultivation media. A) Stereoselective R-alcohol dehydrogenase (ADH) from Lactobacillus produced by E. coli RB791 [pQE30:Adh]; B) Protein disulphide isomerase A-domain (PDI) produced by E. coli BL21(DE3)pLysS [pET23:pdi]; C) Multifunctional enzyme type 2 of Drosophila produced by E. coli BL21(DE3) [pPal7:Dm_MFE-2]. In each sub-picture (A, B, C) soluble proteins are shown on the left and total proteins on the right. The upper and lower parts of the pictures present proteins after 6 h and 24 h induction, respectively. To facilitate direct comparison between different cultivation media, all samples were diluted to equal cell concentration before cell lysis and loading on gel. EB = EnBase Flo medium, MSM = Mineral Salt Medium, LB = Luria Bertani, TB = Terrific Broth. Target protein is indicated by arrows.

Figure 3

Recombinant protein production in 50 ml cultures in 500 ml shake flasks using different cultivation media. A) Stereoselective R-alcohol dehydrogenase (ADH) from Lactobacillus produced by E. coli RB791 [pQE30:Adh]; B) Multifunctional enzyme type 2 of Drosophila produced by E. coli BL21(DE3) [pPal7:Dm_MFE-2]. Each sub-picture presents soluble and total proteins after 6 h and 24 h induction. To facilitate direct comparison between different cultivation media, all samples were diluted to equal cell concentration before cell lysis and loading on gel. EB = EnBase Flo medium, MSM = Mineral Salt Medium, LB = Luria Bertani, TB = Terrific Broth. Target protein is indicated by arrows.

Figure 4

A schematic picture of the EnBase^® Flo cultivation principle. Phase 1 = controlled cultivation in mineral salt medium with a low concentration of complex additives (peptones, yeast extract). Phase 2 = induction with IPTG and addition of complex nutrients (booster mixture). Phase 2a of increased growth rate is followed by Phase 2b of low growth rate and slow production of soluble protein.