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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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ATPase activity of yeast-secreted human BiP. The amount of released phosphate by 1 μg of either bacterial or yeast-derived human BiP was determined after incubation at 25°C for 75 min. with a non-radioactive procedure. Values are the mean of three separate experiments with an error bar representing SD.
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Figure 8: ATPase activity of yeast-secreted human BiP. The amount of released phosphate by 1 μg of either bacterial or yeast-derived human BiP was determined after incubation at 25°C for 75 min. with a non-radioactive procedure. Values are the mean of three separate experiments with an error bar representing SD.

Mentions: ATPase activity of yeast-expressed human BiP protein was measured and compared with commercially available E. coli-derived human BiP using non-radioactive ATPase assay as described in Methods. This test is commonly used to assess activity of the BiP protein[12,31]. As it is evident in Figure 8, both proteins exhibited ATPase activity, but amount of liberated phosphate (μM/hr/μg protein) by yeast-derived BiP was 3-fold larger than that of bacteria-derived protein (~6.3 μM compared to ~2.1 μM) showing a 3-fold higher activity of the yeast-derived protein. The amount of phosphate liberated by E. coli-produced recombinant BiP in our experiments corresponds to that declared by manufacturer, validating our results. Such difference in activity between yeast- and bacteria-derived BiP proteins may be explained by the fact that yeast-secreted human BiP undergoes protein quality control throughout the yeast secretion pathway, which allows secretion of only correctly folded proteins. Meanwhile, E. coli does not have the ER, and the synthesis of recombinant human BiP is performed in different intracellular environment that may be less suitable for the proper maturation of the protein. Similar ratio of ATPase activity comparing native and E. coli-derived recombinant BiP protein was observed earlier[16], where recombinant hamster BiP protein showed only 30% activity of the native bovine liver BiP. Also, as we reported earlier[20], the yeast-secreted human ERp57 protein catalyzed the reduction of insulin in faster rate than recombinant human ERp57 from E. coli. Evidently higher activity of S. cerevisiae-secreted human BiP and ERp57 proteins compared to E. coli-derived recombinant analogues demonstrates that yeast is superior host for the production of BiP and other human ER chaperones.


Generation of human ER chaperone BiP in yeast Saccharomyces cerevisiae.

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

ATPase activity of yeast-secreted human BiP. The amount of released phosphate by 1 μg of either bacterial or yeast-derived human BiP was determined after incubation at 25°C for 75 min. with a non-radioactive procedure. Values are the mean of three separate experiments with an error bar representing SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: ATPase activity of yeast-secreted human BiP. The amount of released phosphate by 1 μg of either bacterial or yeast-derived human BiP was determined after incubation at 25°C for 75 min. with a non-radioactive procedure. Values are the mean of three separate experiments with an error bar representing SD.
Mentions: ATPase activity of yeast-expressed human BiP protein was measured and compared with commercially available E. coli-derived human BiP using non-radioactive ATPase assay as described in Methods. This test is commonly used to assess activity of the BiP protein[12,31]. As it is evident in Figure 8, both proteins exhibited ATPase activity, but amount of liberated phosphate (μM/hr/μg protein) by yeast-derived BiP was 3-fold larger than that of bacteria-derived protein (~6.3 μM compared to ~2.1 μM) showing a 3-fold higher activity of the yeast-derived protein. The amount of phosphate liberated by E. coli-produced recombinant BiP in our experiments corresponds to that declared by manufacturer, validating our results. Such difference in activity between yeast- and bacteria-derived BiP proteins may be explained by the fact that yeast-secreted human BiP undergoes protein quality control throughout the yeast secretion pathway, which allows secretion of only correctly folded proteins. Meanwhile, E. coli does not have the ER, and the synthesis of recombinant human BiP is performed in different intracellular environment that may be less suitable for the proper maturation of the protein. Similar ratio of ATPase activity comparing native and E. coli-derived recombinant BiP protein was observed earlier[16], where recombinant hamster BiP protein showed only 30% activity of the native bovine liver BiP. Also, as we reported earlier[20], the yeast-secreted human ERp57 protein catalyzed the reduction of insulin in faster rate than recombinant human ERp57 from E. coli. Evidently higher activity of S. cerevisiae-secreted human BiP and ERp57 proteins compared to E. coli-derived recombinant analogues demonstrates that yeast is superior host for the production of BiP and other human ER chaperones.

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