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Generation of human ER chaperone BiP in yeast Saccharomyces cerevisiae.

Čiplys E, Aučynaitė A, Slibinskas R - Microb. Cell Fact. (2014)

Bottom Line: Expression of a full-length human BiP precursor in S. cerevisiae resulted in a high-level secretion of mature recombinant protein into the culture medium.Consequently, resulting recombinant BiP protein corresponds accurately to native analogue.The ability to produce large quantities of native recombinant human BiP in yeast expression system should accelerate the analysis and application of this important protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: Vilnius University Institute of Biotechnology, V,A, Graiciuno 8, Vilnius LT-02241, Lithuania. evaldas.ciplys@bti.vu.lt.

ABSTRACT

Background: Human BiP is traditionally regarded as a major endoplasmic reticulum (ER) chaperone performing a number of well-described functions in the ER. In recent years it was well established that this molecule can also be located in other cell organelles and compartments, on the cell surface or be secreted. Also novel functions were assigned to this protein. Importantly, BiP protein appears to be involved in cancer and rheumatoid arthritis progression, autoimmune inflammation and tissue damage, and thus could potentially be used for therapeutic purposes. In addition, a growing body of evidence indicates BiP as a new therapeutic target for the treatment of neurodegenerative diseases. Increasing importance of this protein and its involvement in critical human diseases demands new source of high quality native recombinant human BiP for further studies and potential application. Here we introduce yeast Saccharomyces cerevisiae as a host for the generation of human BiP protein.

Results: Expression of a full-length human BiP precursor in S. cerevisiae resulted in a high-level secretion of mature recombinant protein into the culture medium. The newly discovered ability of the yeast cells to recognize, correctly process the native signal sequence of human BiP and secrete this protein into the growth media allowed simple one-step purification of highly pure recombinant BiP protein with yields reaching 10 mg/L. Data presented in this study shows that secreted recombinant human BiP possesses native amino acid sequence and structural integrity, is biologically active and without yeast-derived modifications. Strikingly, ATPase activity of yeast-derived human BiP protein exceeded the activity of E. coli-derived recombinant human BiP by a 3-fold.

Conclusions: S. cerevisiae is able to correctly process and secrete human BiP protein. Consequently, resulting recombinant BiP protein corresponds accurately to native analogue. The ability to produce large quantities of native recombinant human BiP in yeast expression system should accelerate the analysis and application of this important protein.

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Evaluation of amount of intracellular human BiP protein in yeast S. cerevisiae cells. (A) SDS–PAGE of crude yeast lysates and indicated amounts of purified BiP; (B) Western blot using polyclonal antibodies against human BiP. M – prestained protein ladder (ThermoScientific, cat. no. 26618). pFDC and pFDC-hBiP – crude lysates (10 μg of whole cell protein in each lane) of yeast cells transformed with pFDC vector and pFDC-hBiP plasmid, respectively. 50, 75, 100, 150, 200, 300, 400 – amounts in nanograms of purified secreted human BiP protein loaded on gel.
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Figure 9: Evaluation of amount of intracellular human BiP protein in yeast S. cerevisiae cells. (A) SDS–PAGE of crude yeast lysates and indicated amounts of purified BiP; (B) Western blot using polyclonal antibodies against human BiP. M – prestained protein ladder (ThermoScientific, cat. no. 26618). pFDC and pFDC-hBiP – crude lysates (10 μg of whole cell protein in each lane) of yeast cells transformed with pFDC vector and pFDC-hBiP plasmid, respectively. 50, 75, 100, 150, 200, 300, 400 – amounts in nanograms of purified secreted human BiP protein loaded on gel.

Mentions: As it was mentioned, human BiP protein was expressed previously in S. cerevisiae and was found inside the cell[18]. Here we have shown that this protein is also secreted. To evaluate the efficiency of secretion of human BiP protein driven by its native signal sequence, we compared amounts of intracellular and secreted BiP protein. SDS–PAGE analysis of crude lysates harbouring pFDC-BiP plasmid revealed clear additional band of recombinant human BiP compared to control cells carrying pFDC vector (Figure 9A, lanes pFDC and pFDC-hBiP). Also, quantitative Western blot using antibodies against human BiP protein was performed (Figure 9B). Densitometric analysis of both SDS-PAGE gel and Western blot showed that intracellular BiP constituted for approx. 0.9% of total cell protein. Evaluation of amount of proteins according to cell biomass produced from 1 L of culture revealed that approx. 70% of BiP was expressed internally (approx. 35 mg) and 30% was secreted into the culture medium (approx. 15 mg). Thus, secretion efficiency of human BiP protein in yeast S. cerevisiae cells is slightly higher than that of human ERp57 protein we reported earlier[20].


Generation of human ER chaperone BiP in yeast Saccharomyces cerevisiae.

Čiplys E, Aučynaitė A, Slibinskas R - Microb. Cell Fact. (2014)

Evaluation of amount of intracellular human BiP protein in yeast S. cerevisiae cells. (A) SDS–PAGE of crude yeast lysates and indicated amounts of purified BiP; (B) Western blot using polyclonal antibodies against human BiP. M – prestained protein ladder (ThermoScientific, cat. no. 26618). pFDC and pFDC-hBiP – crude lysates (10 μg of whole cell protein in each lane) of yeast cells transformed with pFDC vector and pFDC-hBiP plasmid, respectively. 50, 75, 100, 150, 200, 300, 400 – amounts in nanograms of purified secreted human BiP protein loaded on gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3926315&req=5

Figure 9: Evaluation of amount of intracellular human BiP protein in yeast S. cerevisiae cells. (A) SDS–PAGE of crude yeast lysates and indicated amounts of purified BiP; (B) Western blot using polyclonal antibodies against human BiP. M – prestained protein ladder (ThermoScientific, cat. no. 26618). pFDC and pFDC-hBiP – crude lysates (10 μg of whole cell protein in each lane) of yeast cells transformed with pFDC vector and pFDC-hBiP plasmid, respectively. 50, 75, 100, 150, 200, 300, 400 – amounts in nanograms of purified secreted human BiP protein loaded on gel.
Mentions: As it was mentioned, human BiP protein was expressed previously in S. cerevisiae and was found inside the cell[18]. Here we have shown that this protein is also secreted. To evaluate the efficiency of secretion of human BiP protein driven by its native signal sequence, we compared amounts of intracellular and secreted BiP protein. SDS–PAGE analysis of crude lysates harbouring pFDC-BiP plasmid revealed clear additional band of recombinant human BiP compared to control cells carrying pFDC vector (Figure 9A, lanes pFDC and pFDC-hBiP). Also, quantitative Western blot using antibodies against human BiP protein was performed (Figure 9B). Densitometric analysis of both SDS-PAGE gel and Western blot showed that intracellular BiP constituted for approx. 0.9% of total cell protein. Evaluation of amount of proteins according to cell biomass produced from 1 L of culture revealed that approx. 70% of BiP was expressed internally (approx. 35 mg) and 30% was secreted into the culture medium (approx. 15 mg). Thus, secretion efficiency of human BiP protein in yeast S. cerevisiae cells is slightly higher than that of human ERp57 protein we reported earlier[20].

Bottom Line: Expression of a full-length human BiP precursor in S. cerevisiae resulted in a high-level secretion of mature recombinant protein into the culture medium.Consequently, resulting recombinant BiP protein corresponds accurately to native analogue.The ability to produce large quantities of native recombinant human BiP in yeast expression system should accelerate the analysis and application of this important protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: Vilnius University Institute of Biotechnology, V,A, Graiciuno 8, Vilnius LT-02241, Lithuania. evaldas.ciplys@bti.vu.lt.

ABSTRACT

Background: Human BiP is traditionally regarded as a major endoplasmic reticulum (ER) chaperone performing a number of well-described functions in the ER. In recent years it was well established that this molecule can also be located in other cell organelles and compartments, on the cell surface or be secreted. Also novel functions were assigned to this protein. Importantly, BiP protein appears to be involved in cancer and rheumatoid arthritis progression, autoimmune inflammation and tissue damage, and thus could potentially be used for therapeutic purposes. In addition, a growing body of evidence indicates BiP as a new therapeutic target for the treatment of neurodegenerative diseases. Increasing importance of this protein and its involvement in critical human diseases demands new source of high quality native recombinant human BiP for further studies and potential application. Here we introduce yeast Saccharomyces cerevisiae as a host for the generation of human BiP protein.

Results: Expression of a full-length human BiP precursor in S. cerevisiae resulted in a high-level secretion of mature recombinant protein into the culture medium. The newly discovered ability of the yeast cells to recognize, correctly process the native signal sequence of human BiP and secrete this protein into the growth media allowed simple one-step purification of highly pure recombinant BiP protein with yields reaching 10 mg/L. Data presented in this study shows that secreted recombinant human BiP possesses native amino acid sequence and structural integrity, is biologically active and without yeast-derived modifications. Strikingly, ATPase activity of yeast-derived human BiP protein exceeded the activity of E. coli-derived recombinant human BiP by a 3-fold.

Conclusions: S. cerevisiae is able to correctly process and secrete human BiP protein. Consequently, resulting recombinant BiP protein corresponds accurately to native analogue. The ability to produce large quantities of native recombinant human BiP in yeast expression system should accelerate the analysis and application of this important protein.

Show MeSH
Related in: MedlinePlus