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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

Partial proteolysis of recombinant BiP with proteinase K in the presence of nucleotides. M – prestained protein ladder (ThermoScientific, cat. no. 26616). Undigested recombinant BiP (A), or digested with proteinase K without nucleotides (B), in the presence of 100 μM ATP (C) or 100 μM ADP (D).
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Figure 7: Partial proteolysis of recombinant BiP with proteinase K in the presence of nucleotides. M – prestained protein ladder (ThermoScientific, cat. no. 26616). Undigested recombinant BiP (A), or digested with proteinase K without nucleotides (B), in the presence of 100 μM ATP (C) or 100 μM ADP (D).

Mentions: To assess the quality of yeast-derived human BiP protein, folding state and activity were determined. Correct folding was evaluated by partial proteolysis of recombinant human BiP protein. Partial protease digestion is often used as a measure of the structural integrity of BiP protein, because HSP70 proteins produce very distinctive proteolytic patterns when digested in the presence of nucleotides. ATP protects 60- and 44-kDa fragments from digestion, whereas ADP protects only the 44-kDa fragment[14,30]. S. cerevisiae-secreted human BiP protein was digested with proteinase K in the presence of ATP or ADP and analyzed by SDS-PAGE as described in Methods (Figure 7). Yeast derived human BiP protein bound ATP, and this protected a ~60 kDa fragment from proteolysis by proteinase K (Figure 7 lane C), whereas binding of ADP protected slightly larger amount of ~44 kDa fragment compared to the samples without added nucleotides or with added ATP (Figure 7 lane D compared to lanes B and C). Similar data was previously used to demonstrate correct folding of both native canine[30] and E. coli-expressed recombinant human BiP proteins[14].


Generation of human ER chaperone BiP in yeast Saccharomyces cerevisiae.

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

Partial proteolysis of recombinant BiP with proteinase K in the presence of nucleotides. M – prestained protein ladder (ThermoScientific, cat. no. 26616). Undigested recombinant BiP (A), or digested with proteinase K without nucleotides (B), in the presence of 100 μM ATP (C) or 100 μM ADP (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Partial proteolysis of recombinant BiP with proteinase K in the presence of nucleotides. M – prestained protein ladder (ThermoScientific, cat. no. 26616). Undigested recombinant BiP (A), or digested with proteinase K without nucleotides (B), in the presence of 100 μM ATP (C) or 100 μM ADP (D).
Mentions: To assess the quality of yeast-derived human BiP protein, folding state and activity were determined. Correct folding was evaluated by partial proteolysis of recombinant human BiP protein. Partial protease digestion is often used as a measure of the structural integrity of BiP protein, because HSP70 proteins produce very distinctive proteolytic patterns when digested in the presence of nucleotides. ATP protects 60- and 44-kDa fragments from digestion, whereas ADP protects only the 44-kDa fragment[14,30]. S. cerevisiae-secreted human BiP protein was digested with proteinase K in the presence of ATP or ADP and analyzed by SDS-PAGE as described in Methods (Figure 7). Yeast derived human BiP protein bound ATP, and this protected a ~60 kDa fragment from proteolysis by proteinase K (Figure 7 lane C), whereas binding of ADP protected slightly larger amount of ~44 kDa fragment compared to the samples without added nucleotides or with added ATP (Figure 7 lane D compared to lanes B and C). Similar data was previously used to demonstrate correct folding of both native canine[30] and E. coli-expressed recombinant human BiP proteins[14].

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