- Open Access
Generation of human ER chaperone BiP in yeast Saccharomyces cerevisiae
© Čiplys et al.; licensee BioMed Central Ltd. 2014
Received: 8 October 2013
Accepted: 8 February 2014
Published: 11 February 2014
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.
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.
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.
Human BiP (immunoglobulin heavy chain-binding protein), also known as GRP78, is an essential ER resident Hsp70 family chaperone. Its functions in the ER are thoroughly studied and well described (reviewed in). BiP is a major chaperone and most abundant protein in the ER, it plays a role in protein transport into the ER, folding, assembly, export and degradation, signal transduction and calcium homeostasis. Therefore, BiP is a central regulator of ER homeostasis and essential for embryonic cell growth and pluripotent cell survival. Over the last decade BiP protein has attracted even more attention due to its newly discovered functions, localization in the cell, involvement in important human diseases and potential therapeutic applications. Recently it was recognized that in specific cell types or when subjected to stress, BiP can be located in cell compartments outside the ER, including the cell surface, the cytoplasm, the mitochondria and the nucleus. Also secretion into the extracellular space, where it affects the cell growth and signalling, was observed (reviewed in).
Particularly intriguing is BiP involvement in progression of critical human diseases, such as cancer and neurodegenerative disorders. Current studies have established that BiP plays crucial and pleiotropic role in cancer progression (reviewed in[3, 5]). It is overexpressed and translocated onto the cell surface in most cancer cells, where it regulates cell survival and proliferation, tumor progression and angiogenesis, and protects cancer cells against the adverse hypoxic and nutrient-deprived microenvironment. Naturally, targeting BiP sensitizes cancer cells to therapy. Furthermore, preferential expression of BiP on the surface of tumor cells but not in normal organs suggests that surface BiP can serve both as a target as well as a mediator for cancer-specific therapy. Recently BiP was suggested as a therapeutic target for neurodegenerative disorders (reviewed in). Most neurodegenerative disorders are characterized by activation of the UPR and altered expression and activity of BiP in ageing cells, raising the question whether the lack of BiP could be a predisposing factor for many neurodegenerative disorders. In some cases endogenous overexpression of BiP was shown to have anti-apoptotic and neuroprotective effects in mice and rat models[9–11]. Taking together, these discoveries change the paradigm on BiP functions and suggest novel therapeutic approaches targeting this protein.
For further research and studies of the potential application of this protein, high quality and widely affordable recombinant human BiP protein is needed. Currently, Escherichia coli is the host of choice for the production of recombinant human BiP protein using various purification techniques[12–16], but resulting protein is less active than native analogue and yields are low[16, 17]. To the best of our knowledge, this work is the first to report yeast Saccharomyces cerevisiae as a host for the generation of human BiP protein. In our previous study, we inserted different human ER chaperones into the yeast to facilitate impaired maturation of recombinant Measles virus hemagglutinin[18, 19]. Even though considerable amount of recombinant human BiP was found to be localized in the yeast ER, subsequent studies revealed that the protein was also secreted outside the yeast cell, similarly to another human ER chaperone ERp57. Here we report how this discovery allows simple and cost-effective purification of large amounts of active human BiP. Our studies revealed that the secretion of human BiP chaperone by yeast cells is mediated by correctly processed native signal sequence of BiP protein. Analysis of the yeast-derived human BiP showed that this protein highly resembles its native analogue. ATPase assay demonstrated that yeast-secreted recombinant protein is 3-fold more active when compared to recombinant BiP purified from E. coli. In conclusion, yeast S. cerevisiae is an excellent host for the production of native recombinant BiP protein.
Results and discussion
Expression and purification of human BiP protein
Molecular weight, processing and oligomerization of recombinant human BiP protein
Taken together, our data indicates that yeast-secreted recombinant human BiP exactly corresponds to the active monomeric form of native mature human BiP protein. It is correctly processed and does not carry any modifications or additional amino acid residues as it is often the case when using S. cerevisiae prepro α mating factor signal sequence for the secretion of recombinant proteins, because of the inefficient cleavage of Ste13 peptidase. It also suggests that translocon machinery and signal peptide peptidase complex of yeast and human cells are compatible, because native signal sequence of human BiP protein is recognized and correctly cleaved in yeast cells. This allows translocation of recombinant protein into the ER followed by unexpected secretion outside the yeast cells. Secretion of human BiP protein by yeast is an interesting phenomenon, especially considering that signal sequence of BiP protein serves as a signal for translocation of protein into the ER, but not as a secretion signal. This finding not only allows simple generation of native human BiP protein in yeast cells, but also might serve as a convenient model to study mechanism of protein retention in the ER. As we reported earlier, human ERp57 protein is also secreted by yeast cells with the intact ER retention signal. Replacing ER retention signals of both BiP and ERp57 to yeast preferable ER retention signal HDEL did not suppress the secretion of those proteins (our unpublished data). Also, overload of the yeast ER retrieval machinery as the reason for secretion of human ERp57 and BiP proteins can be omitted, because overexpression of yeast Kar2 protein with native HDEL ER retrieval sequence using the same pFDC vector did not lead to the secretion of this protein (Additional file1: data 1). These results indicate that the retention of ER luminal proteins is complicated and still unsolved mechanism, which does not strictly depend only on HDEL/KDEL sequences, but is likely a combination of several factors. Observed secretion of human ER luminal proteins ERp57 and BiP by yeast cells can serve as a more convenient model to study this fundamental issue than the full genome-wide screening of yeast Kar2p secretion mutants.
Conformation of yeast-secreted human BiP
ATP-ase activity of yeast-derived human BiP protein
Secretion efficiency of human BiP in yeast
Potential applicability of the results
This study revealed a new potential of the yeast to efficiently produce native recombinant human ER chaperones in the secreted form. It should be noted that a high-level secretion of these proteins by using their native signal sequences in yeast expression system is quite unusual. It is known that some secreted human proteins may be secreted by yeast cells using native secretion signal sequences[32, 33], with a very few examples of a high-level secretion as in the case of human serum albumin in P. pastoris. However, it may be expected that secreted human proteins will also be secreted in yeast cells using the same signal sequence. In contrast, the secretion of intracellular human proteins, such as ER-resident chaperones, is not expected when expressed in yeast. Moreover, the secretion level of human ER chaperones in yeast is unexpectedly high and allows efficient production of correctly processed recombinant products. As it was mentioned, in some cases the native human BiP was shown to be directed to the cell surface or secreted outside the cell where it is involved in a multitude of biological processes. Possible therapeutic applications of BiP are mostly related to the extracellular protein form. For example, BiP is considered as a therapeutic agent of the third generation of biologics for immunological diseases such as rheumatoid arthritis. It was shown that intravenously injected recombinant BiP is able to suppress arthritis and tissue inflammation in mice[35, 36]. Yeast-secreted recombinant protein may be advantageous in such applications, because it is generated in the same way as the native extracellular BiP and corresponds to the native analog insofar as possible. Furthermore, yeast-derived heterologous proteins are free of toxic contaminations and are excellent tools for developing biopharmaceuticals, because S. cerevisiae is acknowledged as GRAS (generally regarded as safe) organism. Therefore, secretory expression of native recombinant human BiP in yeast could be exploited for efficient and safe production of potential therapeutic agent.
Here we introduce the yeast S. cerevisiae as an excellent host for the generation of active human BiP protein. In this study we present evidence that yeast holds several key advantages over E. coli cells currently used for the synthesis of recombinant BiP protein: (a) newly discovered ability of yeast cells to recognize and process the native signal sequence of human BiP and their inability to retain it in the ER leads to secretion of this protein; (b) secretion of the protein allows simple and cost-effective one-step purification; (c) yeast-secreted human BiP exactly corresponds to the native protein; (d) yeast-derived human BiP is correctly folded and three-fold more active than recombinant BiP produced in E. coli. Considering the fact that growing amount of data associates BiP protein with critical human diseases and indicates this protein as a potential therapeutic target or agent, the ability of yeast cells to produce large amounts of native recombinant BiP protein might be invaluable. Also, secretion of this ER luminal human protein by yeast cells might serve as a convenient model to study retention of ER luminal proteins in the ER in general.
Construction of yeast vector for expression of human BiP
All DNA manipulations were performed according to standard procedures. Recombinant plasmids were amplified in E. coli DH5αF’ cells. Human BiP coding gene (HSPA5, GenBank:AF216292) was cloned under the control of constitutive yeast PGK1 gene promoter in pFDC vector, yielding pFDC-BiP plasmid (Figure 1), as it was described previously. Briefly, cDNA encoding full-length human BiP protein precursor was amplified from commercial human adult liver cDNA library (Clontech) by PCR using specific oligonucleotide primers, digested with restriction endonuclease XbaI and cloned into yeast expression vector pFDC. Cloned HSPA5 gene sequence was verified by DNA sequencing and generated plasmid pFDC-BiP was used for the transformation of yeast S. cerevisiae cells.
Yeast strain, medium, transformation and cultivation
S. cerevisiae strain AH22 MATa leu2 his4 was used for expression experiments. Transformation of S. cerevisiae cells was performed by conventional LiCl method. The selection of transformants resistant to formaldehyde was carried out on YEPD (yeast extract 1%, peptone 2%, dextrose 2%) agar supplemented with 4 mM formaldehyde. S. cerevisiae transformants were grown in YEPD medium supplemented with 4 mM formaldehyde.
Protein expression and purification
Yeast cells carrying human HSPA5 gene were grown for 36 h in YEPD medium. Cells were separated from the medium by centrifugation at 2000 g for 10 min. Yeast growth medium was further prefiltered through qualitative filter paper (VWR, cat. No. 516–0812) with subsequent microfiltration through filters with pore size of 1.6 μM (SartoriusStedim Biotech, cat. no. FT-3-1101-047), 0.45 μM (SartoriusStedim Biotech, cat. no. 15406–47) and 0.2 μM (SartoriusStedim Biotech, cat.n o. 15407-47-MIN). After microfiltration, proteins were concentrated and transferred into the binding buffer (20 mM HEPES, 50 mM NaCl, 10 mM MgCl2, pH 8.0) through tangential ultrafiltration using cassettes with 50 kDa cut-off membranes (SartoriusStedim Biotech, cat. no.VF20P3). Further, proteins were mixed with 6-AH-ATP-Agarose (Jena Bioscience, cat. no. AC-129 L) equilibrated in the same buffer and incubated for 2–3 hours at 4°C in batch format. Unbound proteins were removed by washing the resin with 20 column volumes of binding buffer while bound proteins were eluted with equal column volume of elution buffer (20 mM HEPES, 50 mM NaCl, 10 mM MgCl2, 5 mM ATP, pH 7.5). Elution fractions were analyzed by SDS-PAGE. Three subsequent elution fractions showed ~ 95% pure human GRP78/BiP protein. These fractions were pooled and dialysed against ATPase buffer (50 mM HEPES, 50 mM NaCl, 2 mM MgCl2, pH 6.8).
Preparation of crude yeast lysates, SDS-PAGE and Western blotting were performed exactly as described previously.
Partial proteolysis of recombinant BiP in the presence of nucleotides
Partial proteolysis of yeast derived human BiP with proteinase K was performed as described by Wei and Hendershot. 65-μl reactions were assembled that contained 10 μg of recombinant BiP, 2 μg of proteinase K (or similar volume of buffer for control), and 100 μM ATP or ADP in the ATPase buffer (50 mM HEPES, 50 mM NaCl, 2 mM MgCl2, pH 6.8). After incubation at 37°C for 25 min., the reaction was stopped by adding 10 μl of 1 mg/ml phenylmethylsulfonylfluoride and incubating it on ice for 30 min. The digested recombinant BiP was then analyzed by SDS-PAGE.
Non-radioactive ATPase assay was performed as described previously. Reactions were performed in 50 μl volumes as follows: 1 μg of recombinant BiP protein (or equal volume of buffer for negative control) with 20 mM KCl and 20 μM ATP in ATPase buffer (50 mM HEPES, pH 6.8, 50 mM NaCl, 2 mM MgCl2) was incubated at 25°C for 75 min. Concentration of the phospate liberated from ATP was measured by spectrofotometer (TECAN Infinite 200, wave length 620 nm) using Malachite Green Phosphate Assay Kit (Cayman Chemical, cat. no. 10009325) according to manufacturers’ recommendations.
Purified recombinant BiP was mixed in equal volumes with sample buffer (0.01% Bromophenol Blue and 20% glycerol in TBE buffer (90 mM Tris, 90 mM Boric acid, 2 mM EDTA, pH 8)) and loaded onto 10% polyacrylamide gels. Gels were run in TBE buffer in 4°C at 100 V and 30 mA for 8–10 hours. After electrophoresis gels were stained with Coomassie brilliant blue R-250.
Other methods and materials
N terminus sequencing of yeast secreted human BiP protein by Edman degradation was performed by AltaBioscience.
The molecular mass of protein was measured by electrospray mass spectrometry using Agilent Q-TOF 6520 mass spectrometer.
Protein concentrations were determined by Roti-Nanoquant Protein-assay (Carl Roth Gmbh., cat. no. K880).
Densitometric analysis of SDS-PAGE gels and Western blots, scanned with ImageSanner III (GE Healthcare) was performed with ImageQuant TL (GE Healthcare) software using default settings.
Precipitation of proteins from yeast growth medium for SDS-PAGE analysis was performed based on a defined methanol-chloroform-water mixture, as described earlier.
Recombinant human BiP protein purified from E. coli was purchased from StressMarq Biosciences Inc. (cat. no. SPR-107A).
Rabbit polyclonal antibodies against human BiP protein were purchased from Abcam (cat. no. ab21685).
This work was partially supported by UAB Baltymas. We thank Dr. Z. Liutkevičiūtė (Vilnius University, Insitute of Biotechnology) for performing ESI-MS experiments and Dr. R. Ražanskas (Vilnius University, Insitute of Biotechnology) for revision of manuscript.
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