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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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Related in: MedlinePlus

Native PAGE of recombinant human BiP. 5 μg of purified recombinant human BiP was loaded on gel. BSA was loaded as molecular weight marker.
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Figure 6: Native PAGE of recombinant human BiP. 5 μg of purified recombinant human BiP was loaded on gel. BSA was loaded as molecular weight marker.

Mentions: The molecular mass of S. cerevisiae-secreted BiP protein was measured by electrospray mass spectrometry (ESI-MS) using Agilent Q-TOF 6520 mass spectrometer. ESI-MS of a whole S. cerevisiae-derived recombinant BiP protein molecule showed a molecular mass of 70478.39 Da, which exactly corresponds to theoretically predicted mass of mature human BiP (19–654 aa) polypeptide (Figure 5). It indicates that secreted recombinant BiP has no yeast-derived modifications, including phosphorylation and ADP-ribosylation. In mammalian cells free BiP can be reversibly phosphorylated and ADP-ribosylated. Such modifications lead to reversible aggregation and subsequent inactivation of BiP protein[22-26]. Therefore, BiP exists in mammalian cells as an active monomer, less active or inactive dimer, or in inactive oligomeric form[16,17,25]. To determine the oligomerization of the yeast-secreted human BiP, we analyzed it by native PAGE as described in Methods. As it was expected, unmodified yeast-secreted human BiP was present predominantly in monomeric form (Figure 6). For various applications, yeast-secreted recombinant protein might be even preferable over native protein, because majority of the recombinant BiP protein is in active (see below) monomeric form compared to diverse oligomerization of the native mammalian BiP protein.


Generation of human ER chaperone BiP in yeast Saccharomyces cerevisiae.

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

Native PAGE of recombinant human BiP. 5 μg of purified recombinant human BiP was loaded on gel. BSA was loaded as molecular weight marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Native PAGE of recombinant human BiP. 5 μg of purified recombinant human BiP was loaded on gel. BSA was loaded as molecular weight marker.
Mentions: The molecular mass of S. cerevisiae-secreted BiP protein was measured by electrospray mass spectrometry (ESI-MS) using Agilent Q-TOF 6520 mass spectrometer. ESI-MS of a whole S. cerevisiae-derived recombinant BiP protein molecule showed a molecular mass of 70478.39 Da, which exactly corresponds to theoretically predicted mass of mature human BiP (19–654 aa) polypeptide (Figure 5). It indicates that secreted recombinant BiP has no yeast-derived modifications, including phosphorylation and ADP-ribosylation. In mammalian cells free BiP can be reversibly phosphorylated and ADP-ribosylated. Such modifications lead to reversible aggregation and subsequent inactivation of BiP protein[22-26]. Therefore, BiP exists in mammalian cells as an active monomer, less active or inactive dimer, or in inactive oligomeric form[16,17,25]. To determine the oligomerization of the yeast-secreted human BiP, we analyzed it by native PAGE as described in Methods. As it was expected, unmodified yeast-secreted human BiP was present predominantly in monomeric form (Figure 6). For various applications, yeast-secreted recombinant protein might be even preferable over native protein, because majority of the recombinant BiP protein is in active (see below) monomeric form compared to diverse oligomerization of the native mammalian BiP protein.

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