Design of improved membrane protein production experiments: quantitation of the host response

Abstract

Eukaryotic membrane proteins cannot be produced in a reliable manner for structural analysis. Consequently, researchers still rely on trial-and-error approaches, which most often yield insufficient amounts. This means that membrane protein production is recognized by biologists as the primary bottleneck in contemporary structural genomics programs. Here, we describe a study to examine the reasons for successes and failures in recombinant membrane protein production in yeast, at the level of the host cell, by systematically quantifying cultures in high-performance bioreactors under tightly-defined growth regimes. Our data show that the most rapid growth conditions of those chosen are not the optimal production conditions. Furthermore, the growth phase at which the cells are harvested is critical: We show that it is crucial to grow cells under tightly-controlled conditions and to harvest them prior to glucose exhaustion, just before the diauxic shift. The differences in membrane protein yields that we observe under different culture conditions are not reflected in corresponding changes in mRNA levels of FPS1, but rather can be related to the differential expression of genes involved in membrane protein secretion and yeast cellular physiology.

Figure 1.

Culture profile for a transformant grown aerobically on glucose at 30°C (pH 5). Glucose concentration (⋄), OD₆₀₀ (×) and CO₂ level (○) vs. time is shown for growth under these “normal” conditions. The phase of sampling is indicated with an arrow, and each sample is also given a number for ease of identification.

Figure 2.

Growth rates for cultures defined by base addition. Specific growth rate, μ (h⁻¹) was computed using the amount of base added in two adjacent 20-min segments in the 1.5–7 g/L glucose range. Conditions are as follows: 30°C (pH 5) (squares); 35°C (pH 5) (triangles); 35°C (pH 7) (crosses); 30°C (pH 7) (diamonds); 20°C (pH 5) (circles).

Figure 3.

Yield of membrane-bound Fps1p as a function of growth phase. In subsequent calculations, the amount of membrane-bound Fps1p was quantified from immunoblot using the integration window shown. “-Glc” and “-EtOH” refer to samples extracted during the glucose and ethanol growth phases, respectively, as illustrated in Figure 1 ▶.

Figure 4.

Quantitation of relative Fps1 protein yields. Immunoblots were performed and quantified on both the total (A) and membrane-bound (B) fractions for samples cultured under the five conditions shown, and harvested as indicated in Figure 1 ▶. “-Glc” and “-EtOH” refer to samples extracted during the glucose and ethanol growth phases, respectively. The immunoblot signals were quantified in the ImageGauge program. Either total extract or the membrane-bound fraction isolated from Sample 2 of the 30°C (pH 5) culture was used as an internal standard. All signals were below saturation and related to the signal of the internal standard. Duplicate fermentations (solid bars) were used to calculate the standard error of the mean at 30°C (pH 5) and 35°C (pH 5). For all other conditions, the error (hatched bars) was estimated based on the values for 30°C (pH 5) and 35°C (pH 5). Average values were calculated for total Fps1 protein in both the glucose and ethanol phases to give an indication of yield variation within and between different growth conditions.

Figure 5.

Quantitation of the yield of membrane-bound Fps1p as a function of dry weight and growth phase. The amount of membrane-bound Fps1p was quantified from immunoblot (Fig. 4 ▶) and related to the appropriate dry weight for the time point (point 3 for “3-Glc” and the average of points 5–7 for “Average EtOH”). In all cases, except at 35°C (pH 7), the maximum yield was obtained just prior to the diauxic shift (3-Glc). In the ethanol phase, yields were fairly constant throughout that part of the growth curve, as indicated by the averages given.