Adaptation of Osborne-fractionation

Results

3.2 Optimization of downstream processing

precipitation reagent was kept at 100% to reduce dilution. Ratio was varied from 0.25 to 5 for different lysates. Loss of specific protein is presented in Figure 19.

0%

20%

40%

60%

80%

100%

0 1 2 3 4 5

2-propanol/lysate-ratio calculated and normalized yield of WSP in preextract

HMW-Dx5 HMW-Dy10 LMW6

Figure 19: Osborne-fractionation: Influence of 2-propanol/yeast-lysate-ratio on precipitation of WSPs WSP content in supernatant after precipitation normalized to maximal values

Optimal ratios for the efficient precipitation without loss of recombinant protein were both, very low, below 0.5 and very high, for LMW6 at least 4 and for HMW 1.5.

Low ratios had to be preferred, because of its low 2-propanol-amount. With decreasing alcohol amount the increase in host protein contamination could be observed. A ratio of 0.5 was therefore optimal for precipitation for all WSPs and could be performed for all subunits, even if expressed in one host.

Next parameter, that was investigated, was the percentaged 2-propanol amount in the extraction buffer. To achieve a defined concentration in the mixture, the different lysates were previously precipitated. The normalized yield from the different extraction mixtures are represented in Figure 20. Here it is visible, that there was a strong correlation between HMW-1Dx5 and HMW-1Dy10. LMW6 was extracted in a smaller concentration range and good results could be obtained from 30 to 60% (v/v), but 60% (v/v) was preferred because of the contamination with host proteins at lower amounts. HMW-subunits were extracted at high yields in a broad range (50%). The highest yield could be determined around 40% (v/v) 2-propanol. If extraction buffer was directly given to the lysate, condition changed. Extraction buffer was diluted with lysate and optimal concentration was shifted as represented for LMW6 for 10% (v/v). This extraction method was accompanied by higher 2-propanol requirements and contamination with host cell proteins was increased. Nevertheless, this could be used for the easy detection of WSPs also in ATPS.

Results

0%

20%

40%

60%

80%

100%

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

2-propanol concentration in extraction buffer calculated and normalized yield of WSP in extract

HMW-Dx5 HMW-Dy10 LMW-6

Figure 20: Osborne-fractionation: Influence of 2-propanol concentration on extraction of diff. WSP from yeast

Extraction was performed at different concentrations of 2-propanol in the extraction buffer, quantification was performed by western blot (LMW) and coomassie stained gel (HMW)

The following important parameter was the extraction buffer/precipitate-ratio. Therefore, a defined amount of lysate was precipitated as previous confirmed and extraction buffer volume was varied. Figure 21 gives information about the achieved yields. Here is to be recognized, that, as expected, with increasing volume of extracting agent the extraction yield increased.

Due to the logarithmic process the necessary ratio can be restricted. An increase to more than a factor of three did not appear reasonable, since the additional protein yield and the protein concentration were decreased.

0%

20%

40%

60%

80%

100%

0 1 2 3 4 5

extraction buffer/lysate-ratio calculated and normalized yield of WSP in extract

HMW-Dx5 HMW-Dy10 LMW-6

HMW-subunits showed a higher solubility in the extraction buffer. Lower affinity of LMW6 was accompanied by higher necessary volume-ratio. Factor two seemed to be sufficient in all cases. Higher loss of protein was observed for LMW6 than for the HMW-subunits.

Last step in the purification of WSPs was the precipitation to get rid of the alcohol mixture and to further purify WSPs by removing host cell proteins. As in the previous sections 2-propanol was used at different amounts.

Table 6: Osborne-fractionation: Influence of 2-propanol concentration and temp. on precipitation of diff.

WSP

Quantification was performed by western blot (LMW) and coomassie staining (HMW), normalized to highest value of precipitation of each WSP

Normalized yield of WSPs after precipitation 2-prop. concentration

in mixture HMW-1Dx5 at 4°C HMW-1Dy10 at 4°C LMW6 at 25°C LMW6 at 4°C

50% 11% 12% 2% 100%

60% 20% 26% 22% 92%

70% 92% 97% 34% 99%

80% 100% 100% 46% 88%

90% 95% 93% 100% 68%

95% 44% 33% 97% 60%

Table 6 shows that an increase of 2-propanol-concentration via 100% 2-propanol addition led to higher precipitation rates. HMW-subunit precipitation decreased with exceeding 80%, whereas LMW6 precipitation could be further enhanced at 25°C. Elevation for execution at 4°C could be obtained for lower 2-propanol contents. For effective precipitation all WSPs should be treated with 70% 2-propanol at 4°C.

An interesting observation was made, when HMW-extracts with different content of 2-propanol in extraction buffer were precipitated without further addition of 2-2-propanol. Figure 22 shows the surprising results.

Std M 0 10 20 30 40 50 60 70 80 90%

Figure 22: Osborne-fractionation: Influence of 2-propanol concentration precipitation of HMW from yeast

Gel was Coomassie stained and HMW-subunits were extracted with different 2-propanol concentrations and precipitated at 4°C without further addition of 2-propanol

Results As already shown, the extraction was optimal for HMW-subunits between 20 and 40% (v/v) 2-propanol and 1% DTT. Cooling at 4°C and following centrifugation showed precipitation between extract concentrations of 10 to 30% (v/v). Highest yield with lowest host protein contamination was obtained for 20%. If only HMW has to be separated this procedure would be with lowest 2-propanol usage. And precipitation is initiated only by cooling.

3.2.1.1 Common extraction of HMW- and LMW6-glutenins

The extraction of the different wheat storage proteins significantly differed, but a compromise for extract all WSP in one extraction could be generated without big loss of WSPs.

Extractions in which all four different wheat storage proteins are supposed to be simultaneously isolated were represented through following protocol:

Precipitation:

Add to lysate to 25% (v/v) of a 100% 2-propanol, mix

Centrifugation: 10' at 9000rpm at 4°C (lower speed for easy resuspension) Extraction:

Resuspend pellet with 40-45% (v/v) 2-propanol and 1% (w/v) DTT Extraction for 1 h at 60°C, when appropriate resuspend again Centrifugation: 10' at 100% speed at 40°C (to avoid precipitation) Precipitation:

Shift of concentration of 2-propanol through addition of 100% 2-propanol up to 80 % Centrifugation: 10' at full speed at 4°C (enhance precipitation)

The buffering to lower pH values was accompanied by a lower host cell protein contamination and higher specific protein amount. New extraction method compared to the classical one exhibit lower loss of specific protein during first precipitation, here the removal of host specific proteins was strongly enhanced. This consequently led to lower host cell protein contamination in extract and in last precipitation. As a result, the requirement for chemical compounds (e.g. 2-propanol) could be significantly reduced by the new extraction method.