High-yield production of biologically active recombinant protein in shake flask culture by combination of enzyme-based glucose delivery and increased oxygen transfer

Abstract

This report describes the combined use of an enzyme-based glucose release system (EnBase®) and high-aeration shake flask (Ultra Yield Flask™). The benefit of this combination is demonstrated by over 100-fold improvement in the active yield of recombinant alcohol dehydrogenase expressed in E. coli. Compared to Terrific Broth and ZYM-5052 autoinduction medium, the EnBase system improved yield mainly through increased productivity per cell. Four-fold increase in oxygen transfer by the Ultra Yield Flask contributed to higher cell density with EnBase but not with the other tested media, and consequently the product yield per ml of EnBase culture was further improved.

Figure 1

Ultra Yield Flask with nominal volume of 500 ml.

Figure 2

Cell growth. Cell density by OD₆₀₀ readings for EnBase (A) as well as TB and ZYM-5052 (B) cultures of E. coli RB791[pQE30:adh] in Ultra Yield and Erlenmeyer flasks. One unit of OD₆₀₀ corresponds to dry cell weight of 0.27 g l^-1. Time of inducer addition is indicated by a straight vertical line. In EnBase cultures the Booster tablet was added at the same time with inducer. Note the different scales in figures A and B. EB: EnBase; TB: Terrific Broth; ZYM: ZYM-5052 autoinduction medium; UY: Ultra Yield Flask; Erl: Erlenmeyer flask. Concentrations of glucose-releasing enzyme in EnBase cultures (A) are presented in the legend in U l^-1.

Figure 3

Medium pH. pH in EnBase (A) as well as TB and ZYM-5052 (B) cultures of E. coli RB791[pQE30:adh] in Ultra Yield and Erlenmeyer flasks. Time of inducer addition is indicated by a straight vertical line. In EnBase cultures the Booster tablet was added at the same time with inducer. Note the different time scales in figures A and B. EB: EnBase; TB: Terrific Broth; ZYM: ZYM-5052 autoinduction medium; UY: Ultra Yield Flask; Erl: Erlenmeyer flask. Concentrations of glucose-releasing enzyme in EnBase cultures (A) are presented in the legend in U l^-1.

Figure 4

Adh activity per ml. Volumetric activity (units per ml of culture broth) of the recombinant Adh product expressed in E. coli RB791 in EnBase, TB and ZYM cultures in Ultra Yield and Erlenmeyer flasks. 1 unit is defined as the amount of ADH required for conversion of 1 mM substrate min^-1 at 20°C and pH 7.0. EB: EnBase; TB: Terrific Broth; ZYM: ZYM-5052 autoinduction medium; UY: Ultra Yield Flask; Erl: Erlenmeyer flask. The values 0.6, 1.5, 3 and 6 associated with EnBase cultures on the x axis refer to glucose-releasing enzyme concentrations in U l^-1.

Figure 5

Adh activity per biomass. Specific activity (units per mg of cell dry weight) of the recombinant Adh product expressed in E. coli RB791 in EnBase, TB and ZYM cultures in Ultra Yield and Erlenmeyer flasks. 1 unit is defined as the amount of ADH required for conversion of 1 mM substrate min^-1 at 20°C and pH 7.0. EB: EnBase; TB: Terrific Broth; ZYM: ZYM-5052 autoinduction medium; UY: Ultra Yield Flask; Erl: Erlenmeyer flask. The values 0.6, 1.5, 3 and 6 associated with EnBase cultures on the x axis refer to glucose-releasing enzyme concentrations in U l^-1.

Figure 6

Cell lysates on reducing SDS-PAGE gels. A: soluble protein fraction; B: total protein including both soluble and insoluble fractions. The same dilution was used for all samples, i.e. lysates from equal broth volumes were loaded on the gels, and hence the bands represent protein yield per unit volume of culture broth. EB: EnBase; TB: Terrific Broth; ZYM: ZYM-5052 autoinduction medium; UY: Ultra Yield Flask; Erl: Erlenmeyer flask. The values 0.6, 1.5, 3 and 6 associated with EnBase cultures refer to glucose-releasing enzyme concentrations in U l^-1.