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Yeast Protein Production System Features High Yields and One-Step,,,Purification

ides sufficient thiamine to repress the nmt1 promoter. Expression is induced by harvesting the cells and growing them in EMM broth without thiamine for approximately 18 hours.

Figure 2

The chicken calmodulin gene was inserted into the pESP-1 vector and expressed in S. pombe. Figure 2 shows the cell lysate samples derived from repressed and induced cells containing chicken calmodulin. When cells were grown under induced conditions, the crude lysate shows a dominant band at the size expected for a GST-calmodulin fusion protein (43 kDa) (figure2, lane 4). In the lysate of cells grown under repressed conditions, this band was absent. (Figure 2, lane 1) When the samples were subjected to one-step purification of the GST fusion protein by using GST affinity resin, the induced GST-calmodulin fusion protein was depleted in the glutathione resin flowthrough sample (Figure 2, lane 5) and was eluted to near homogeneity in the glutathione elution fraction (Figure 2, lane 6). No detectable GST fusion protein was observed when lysate derived from repressed cells was subjected to the same purification procedure (Figure 2, lane 3). Figure 3 shows Western blot analysis of the GST-calmodulin fusion protein in cell lysates derived from repressed and induced cells. The fusion protein was not detected in cells when the promoter was repressed but was abundant in cells when the promoter was induced. We have also observed that constitutive, high-level expression of GST fusion proteins is possible by inoculating the cells directly into the induction medium (EMM), which significantly simplifies the overall procedure. However, proteins
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