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Mutant human protein disulfide isomerase assists protein folding in a chaperone-like fashion
Journal of Biotechnology 54 (1997) 105 – 112
Mutant human protein disulfide isomerase assists protein folding in a chaperone-like fashion Yin Gao, Hui Quan, Meiyan Jiang, Yong Dai, Chih-chen Wang * National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, People’s Republic of China Accepted 14 February 1997
Abstract Human protein disulfide isomerase with an extra 10 amino acid residues of AEITRIDPAM at the N-terminal was expressed in E. coli as a soluble protein comprising 20% of total cell proteins, and was purified to near homogeneity through one step of DEAE-Sephacel chromatography. The mutant enzyme, which had the same CD spectrum and comparable disulfide isomerase and thiol-protein oxidoreductase activities with that of the wild type human and bovine protein disulfide isomerases, also showed chaperone-like activity in stimulating the refolding of proteins containing no disulfide bond. The overall yield of the active product is about 20 mg l − 1 culture. © 1997 Elsevier Science B.V. Keywords: Disulfide isomerase; Protein refolding; Chaperone; High-level expression; Purification
1. Introduction It is now generally accepted that the folding and assembly of nascent peptides into functional proteins in most cases require the assistance of other proteins, which can be classified as molecular chaperones and foldases (Gething and Sambrook, 1992). Protein disulfide isomerase (PDI, EC 5.3.4.1), a multifunctional protein of 57 kDa in the lumen of endoplasmic reticulum and one of * Corresponding author. Tel.: +86 10 62022027; fax: + 86 10 62022026; e-mail: [email protected]
the two foldases so far characterized, is involved in protein folding by catalyzing the formation of native disulfide bonds of nascent peptides (Noiva, 1994). In addition to its isomerase activity, it has been suggested (Wang and Tsou, 1993) and demonstrated recently that PDI has a chaperonelike activity in assisting the reactivation upon dilution of guanidine hydrochloride (GuHCl) denatured proteins containing no disulfide bond (Cai et al., 1994; Song and Wang, 1995). Similar results have been reported for disulfide containing proteins (Puig and Gilbert, 1994). In addition, it has been suggested that the essential function of
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Y. Gao et al. / Journal of Biotechnology 54 (1997) 105–112
yeast PDI does not reside in its isomerase activity as a yeast mutant with PDI inactivated by mutation at the active sites is still viable (LaMantia and Lennarz, 1993). However, little is known on the mechanisms underlying the disulfide isomerase and chaperone activities of PDI. Genes for PDI of rat (Gilbert et al., 1991; Laboissiere et al., 1995), murine (Haugejorden et al., 1992), human (Vuori et al., 1992a; Tojo et al., 1994) and yeast (LaMantia and Lennarz, 1993) have been cloned and expressed in E. coli, yeast or insect cells with a wide range of yields from 2–30 mg PDI l − 1 culture. PDI mutants have also been made for studies on relationship of structure and function (LaMantia and Lennarz, 1993; Vuori et al., 1992b,c; Puig et al., 1994; Lyles and Gilbert, 1994; Darby and Creighton, 1995). PDI has been shown to assist refolding and therefore improve the in vitro renaturation yield of recombinant products expressed as inclusion bodies (Buchner et al., 1992; Tang et al., 1994) and to increase the secretion of foreign proteins in yeast cells when PDI itself is overexpressed (Robinson et al., 1994). In order to elucidate the structural basis of the isomerase and chaperone activities of PDI and mechanism of its function, it is desirable to construct a system for expression of PDI with high yield and a simple purification procedure. In this paper we reported the expression of wild type human PDI (rhPDI) and a mutant human PDI with extra 10 amino acid residues at the N-terminal (mhPDI) as soluble cytoplasmic protein in E. coli. The mhPDI was expressed with much higher yield than that for rhPDI without losing any activities of the wild type enzyme, such as disulfide isomerase, thiol-protein oxidoreductase (TPOR) and chaperone activities.
2. Materials and methods
2.1. Construction of expression 6ector pBV-rhPDI and pBV-mhPDI The plasmid pBR322-PDI, containing the full length human PDI cDNA, was generously pro-
vided by Prof K. Kivirikko (Pihlajaniemi et al., 1987). The 5% noncoding region of PDI gene was removed and then subcloned into pBluescript II SK(-) phagemid (Stratagene) to generate the plasmid BS-PDI (Gao and Wang, 1996). The construction of the expression vector pBV-rhPDI is shown in Fig. 1A. The EcoRI/BamHI-digested 1.7-kb pairs fragment from the plasmid, BS-PDI, containing the coding frame of the hPDI was cloned into the expression vector, pBV220 (Zhang et al., 1990). BS%-PDI was derived from the BS-PDI plasmid but with the insert of human PDI cDNA in a reversed direction. The DNA fragment I of 53 bp was synthesized on an Applied Biosystems DNA Synthesizer 381A (Fig. 1B). The DNA fragment II of about 0.4 kb pairs was generated from BS%-PDI with A6aI, mung bean nuclease and PstI sequential digestion. The DNA fragment III was obtained from BS%-PDI after PstI and SalI digestion. For expression of mhPDI, the pBV-mhPDI was constructed with above three DNA fragments and the digested pBV220 with EcoRI, mung bean nuclease and SalI (Fig. 1C).
2.2. Purification of bPDI, mhPDI and rhPDI The bPDI was prepared from bovine liver essentially according to Lambert and Freedman (1983). E. coli DH5a cells bearing pBV-mhPDI were grown for 4–5 h after induction at 42°C and harvested by centrifugation. The cell pellet was resuspended in lysis buffer (0.1 M Tris, pH 8.0, 0.01% lysozyme) and incubated at 30°C for 2–3 h. Ammonium sulfate was added to the supernatant of the lysate to 85% saturation and the precipitate was dissolved in 10 mM phosphate buffer, pH 6.3. After thorough dialysis, it was then purified by DEAE-Sephacel chromatography in the same buffer with a linear gradient of 0.1–0.6 M NaCl. The mhPDI fractions were identified by isomerase activity assay and pooled at 0.4–0.5 M NaCl (Fig. 2A), desalted and lyophilized. The simple procedure for purifying mhPDI is not enough for the purification of rhPDI expressed in E. coli DH5a probably because of its low level expression and the fact that additional chromatography with hy-
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Fig. 1. (A) Construction of the expression vector pBV-rhPDI. (B) The 5% regions and the corresponding amino acid residues at the N-terminal of products of the wild type and the mutant human PDI genes. (C) Construction of the vector pBV-mhPDI for high-level expression of mhPDI.
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Fig. 1. (Cont.)
droxylapatite column and Mono-Q FPLC are employed following DEAE-Sephacel chromatography.
Chaperone-like activity was examined according to Cai et al. (1994).
2.4. CD spectrum determinations 2.3. Acti6ity assay The disulfide isomerase and TPOR activities of wild and mutant PDI preparations were assayed according to Lambert and Freedman (1983).
CD spectrum determinations in the far ultraviolet region from 200–250 nm were carried out with a Jasco J-720 spectropolarimeter.
Y. Gao et al. / Journal of Biotechnology 54 (1997) 105–112
3. Results
Table 1 Enzymatic activities of mhPDI, rhPDI and bPDI
3.1. High-le6el expression in E. coli and purification of mhPDI The rhPDI was expressed with vector pBV220
109
Isomerase (U g−1) TPOR (U g−1)
mhPDI
rhPDI
bPDI
1290 248
930 220
890 230
containing a PRPL promoter in E. coli as a cytoplasmic protein with an unsatisfactory yield. In order to improve the expression level the human PDI cDNA was extended at its 5% region by adding 39 bases. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins of E. coli DH5a cells bearing the mhPDI cDNA as above showed that the mhPDI was expressed as a cytosolic protein constituting about 20% of total cell proteins and 40% of total proteins in the lysate supernatant (Fig. 2B). The preparation of mhPDI with higher than 90% purity was obtained from the supernatant of the cell lysate by ammonium sulfate precipitation and a single step of DEAESephacel chromatography (Fig. 2) with a final yield of about 20 mg l − 1 culture. The final rhPDI preparation is 95% pure.
3.2. Enzyme acti6ities and secondary structure of mhPDI
Fig. 2. Characterization and purification of expressed mhPDI. (A) DEAE-Sephacel chromatography of the lysate supernatant was carried out with a linear gradient of 0.1–0.6 M NaCl in 10 mM phosphate buffer, pH 6.3. The arrow indicates the mhPDI peak. (B) SDS-PAGE was performed using Bio-Rad Mini-Gel system with 10% gel. 1 and 6, Pharmacia HMW marker; 2, total Iysate proteins of DH5a with pBV220; 3, total cell proteins of DH5a bearing pBV-mhPDI; 4, total proteins of lysate supernatant; 5, purified mhPDI after DEAE-Sephacel chromatography.
PDI catalyzes the oxidation, reduction and isomerization of protein disulfides depending on the redox potentials in the reaction medium (Lambert and Freedman, 1983). Ribonuclease with scrambled disulfides and insulin were commonly used as substrates for determinations of isomerase and TPOR activities, respectively (Lambert and Freedman, 1983). Table 1 shows that the isomerase and TPOR activities of purified mhPDI are both in the normal ranges of activities of rhPDI and bPDI preparations. As shown in Fig. 3, the circular dichronism spectrum of mhPDI is almost identical to that of rhPDI and bPDI, suggesting that the N-terminal lengthened enzyme has secondary structures closely similar to that of the native enzyme. The addition of 10 amino acid residues AEITRIDPAM at the N-terminal has no significant effect on either the secondary structure or the enzyme activities.
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3.3. Chaperone-like acti6ity of mhPDI It has been widely accepted that molecular chaperones assist protein folding through binding with folding intermediates to prevent their incorrect interactions resulting in aggregation, so as to favor the productive folding pathway and therefore increase the yield of the native protein. In the system established in this laboratory the reactivation and suppression of aggregation of GuHCl denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) upon dilution in the presence and absence of various PDI preparations were used to measure the chaperone activity of PDI preparations (Cai et al., 1994). Fig. 4A shows that the reactivation of GAPDH increases from 4–18% when the molar ratio of mhPDI to GAPDH (in promoter) is increased from 0–6 with no further increase at higher mhPDI concentrations, which is closely similar to the effect of rhPDI and bPDI on the reactivation of GAPDH. In contrast, bovine serum albumin at a molar ratio of 5 has no effect on the reactivation yield of denatured GAPDH. At the same time the severe aggregation of GAPDH during spontaneous refolding monitored by light scattering was suppressed with increasing
Fig. 4. Chaperone-like activity of PDI preparations. (A) Effect of concentrations of PDI preparations on the reactivation of GuHCl denatured GAPDH at 2.8 mM. Concentrations of rhPDI (), mhPDI () and bPDI ( ) were as indicated. Bovine serum albumin of 14 mM (") was used as control. (B) Effect of concentrations of mhPDI on the aggregation of denatured GAPDH upon dilution as monitored by light scattering. The numbers indicate the molar ratios of mhPDI to GAPDH in promoter.
Fig. 3. The CD spectra of PDI preparations. Protein concentrations were 5.53, 3.96 and 3.74 mM for (—) mhPDI; (- -) bPDI and (···) rhPDI, respectively. Cuvettes of l mm pathlength were used for CD measurements.
concentrations of mhPDI present in the refolding system as shown in Fig. 4B, which is also very similar to the effects of bPDI on the aggregation of GAPDH (Cai et al., 1994). All the above indicate that mhPDI, as well as rhPDI and bPDI, showed chaperone-like activity to suppress aggregation of GAPDH during refolding and thereby assist its correct folding and increase the reactivation yield.
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4. Discussion
Acknowledgements
PDI catalyzes the formation and rearrangement of disulfide bonds of proteins and plays a critical role in nascent peptide folding. In addition the multifunctional PDI displays molecular chaperone activity. However, it is less sure if the enzyme plays different roles either under different conditions or simultaneously. The addition of 30 bp following the ATG start codon at the 5% terminal of the hPDI cDNA with favorable codons of prokaryotic genes designed to enhance translation initiation (Grosjean and Fiers, 1982) does increase the expression level of mhPDI to 20% of the total cell proteins and 40% of the total proteins in the lysate supernatant in contrast to the expression of rhPDI of only a few percent. The sequence between the Shine-Dalgarno region and the start codon has also been extended and changed to A + T rich bases, which may function to increase translation efficiency (Gold et al., 1981). The present study provides a system adequate for the preparation of large amounts of mhPDI with higher than 90% homogeneity from the cell lysate through a simple procedure, ammonium sulfate precipitation followed by one step of DEAE-Sephacel chromatography. The product retains the same enzymatic and chaperone activities as that of the wild type human and bovine PDI, the purification of which often requires a lengthy procedure to give a similar purity. The addition of 10 amino acid residues at the N-terminal has no negative effect on the activities of PDI. The mutant protein appears to be even slightly more active in both the isomerase and the oxidoreductase activities suggesting that the extra stretch of 10 amino acid residues at the N-terminus does not interfere with the structure of either the active sites or the peptide binding sites, as peptide binding site is necessary for both isomerase activity and chaperone activity (Quan et al., 1995; Hayano et al., 1995). Actually the secondary structure of the PDI molecule was also not affected by the N-terminal extension of 10 amino acid residues as shown by CD measurement. Therefore, the mutant PDI can be taken as native PDI in basic studies and applied in protein biotechnology.
We are sincerely grateful to Prof. K. Kivirikko of the University of Oulu for his generous gift of human PDI cDNA and Prof. Y-D. Hou of the Institute of Virology, China Academy of Preventive Medical Science for plasmid pBV220. This work was supported by the Pandeng Project of the Chinese Commission of Science and Technology, China Postdoctoral Science Foundation and Grant No. 39470163 of the China Natural Science Foundation. We thank Prof C.L. Tsou for his continuous encouragement and helpful advice. References Buchner, J., Brinkmann, U. and Pastan, I. (1992) Renaturation of a single-chain immunotoxin facilitated by chaperones and protein disulfide isomerase. Bio/Technology 10, 682 – 685. Cai, H., Wang, C.C. and Tsou, C.L. (1994) Chaperone-like activity of protein disulfide isomerase in the refolding of a protein with no disulfide bonds. J. Biol. Chem. 269, 24550 – 24552. Darby, N.J. and Creighton, T.E. (1995) Functional properties of the individual thioredoxin-like domains of protein disulfide isomerase. Biochemistry 34, 11725 – 11735. Gao, Y. and Wang, C.C. (1996) Expression of human protein disulfide isomerase in Escherichia coli using plasmid pUC18 as an expression vector. Chinese Biochem. J. 12, 237 – 239. Gething, M.J. and Sambrook J. (1992) Protein folding in the cell, Nature 355, 33 – 45. Gilbert, H.F., Kruzel, M.L., Lyles, M.M. and Harper, J.W. (1991) Expression and purification of recombinant rat protein disulfide isomerase from Escherichia coli. Protein Expression Purif. 2, 194 – 198. Gold, L., Pribnow, D., Schneider, T., Shinedling, S., Singer, B.S. and Stormo, G. (1981) Translational initiation in prokaryotes. Annu. Rev. Microbiol. 35, 365 – 403. Grosjean, F. and Fiers, W. (1982) Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes. Gene 18, 199 – 209. Haugejorden, S., Srinivasan, M. and Green, M. (1992) The expression of murine protein disulfide isomerase in Escherichia coli. DNA Cell Biol. 11, 405 – 414. Hayano, T., Hirose, M. and Kikuchi, M. (1995) Protein disulfide isomerase mutant lacking its isomerase activity accelerates protein folding in the cell. FEBS Lett. 377, 505 – 511. Laboissiere, M.C.A., Chivers, P.T. and Raines, R.T. (1995) Production of rat protein disulfide isomerase in Saccharomyces cere6isiae. Protein Expression Purif. 6, 700 – 706.
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LaMantia, M. and Lennarz, W.J. (1993) The essential function of yeast protein disulfide isomerase does not reside in its isomerase activity. Cell 74, 899–908. Lambert, N. and Freedman, R.B. (1983) Structural properties of homogeneous protein disulfide-isomerase from bovine liver purified by a rapid high-yielding procedure. Biochem. J. 213, 225 – 234. Lyles, M.M. and Gilbert, H.F. (1994) Mutations in the thioredoxin sites of protein disulfide isomerase reveal functional nonequivalence of the N- and C-terminal domains. J. Biol. Chem. 269, 30946–30952. Noiva, R. (1994) Enzymatic catalysis of disulfide formation. Protein Expression Purif. 5, 1–13. Pihlajaniemi, T., Helaakoski, T., Tasanen, K., Myllyla¨, R., Huhtala, M-L., Koivu, J. and Kivinkko, K.I. (1987) Molecular cloning of the b-subunit of human prolyl 4-hydroxylase: this subunit and protein disulfide isomerase are products of the same gene. EMBO J. 6, 643–649. Puig, A. and Gilbert, H. (1994) Protein disulfide isomerase exhibits chaperone and anti-chaperone activity in the oxidative refolding of lysozyme, J. Biol Chem. 269, 7764 – 7771. Puig, A., Lyles, M.M., Noiva, R. and Gilbert, H.F. (1994) The role of the thiol/disulfide centers and peptide binding site in the chaperone and antichaperone activities of protein disulfide isomerase. J. Biol. Chem. 269, 19128–19135. Quan, H., Fan, G. and Wang, C.C. (1995) Independence of the chaperone activity of protein disulfide isomerase from its CGHC-active site. J. Biol. Chem. 270, 17078–17080. Robinson, A.S., Hines, V. and Wittrup, K.D. (1994) Protein disulfide isomerase overexpression increases secretion of foreign proteins in Saccharomyces cere6isiae. Bio/Technology 12, 381 – 384.
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Song, J.L. and Wang, C.C. (1995) Chaperone-like activity of protein disulfide isomerase in the refolding of rhodanese. Eur. J. Biochem. 231, 312 – 316. Tang, B., Zhang, S. and Yang, K. (1994) Assisted refolding of recombinant prochymosin with the aid of protein disulfide isomerase. Biochem. J. 301, 17 – 20. Tojo, H., Asano, T., Kato, K., Udaka, S., Horiuchi, R. and Kakinuma, A. (1994) Production of human protein disulfide isomerase in Bacillus bre6is. J. Biotechnol. 33, 55 – 62. Vuori, K., Pihlajaniemi, T., Marttila, M. and Kivirikko, K. (1992a) Characterization of the human prolyl 4-hydroxylase tetramer and its multifunctional protein disulfide isomerase subunit synthesized in a baculovirus expression system. Proc. Natl. Acad. Sci. U.S.A. 89, 7467 – 7470. Vuori, K., Pihlajaniemi, T., Myllyla¨, R. and Kivirikko, K.I. (1992b) Site-directed mutagenesis of human protein disulfide isomerase: effect on the assembly, activity and endoplasmic reticulum retention of human prolyl 4-hydroxylase in Spodopterafugiperda insect cells. EMBO J. 11, 4213 – 4217. Vuori, K., Myllala¨, R., Pihlajaniemi, T. and Kivirikko, K.I. (1992c) Expression and site-directed mutagenesis of human protein disulfide isomerase in Escherichia coli. J. Biol. Chem. 267, 7211 – 7214. Wang, C.C. and Tsou, C. L. (1993) Protein disulfide isomerase is both an enzyme and a chaperone. FASEB J. 7, 1515 – 1517. Zhang, Z.Q., Yao, L.H. and Hou, Y.D. (1990) Construction and application of a high level expression vector containing PR PL promoter, Chinese J. Virol. 6, 111 – 116.
.
Mutant human protein disulfide isomerase assists protein folding in a chaperone-like fashion Yin Gao, Hui Quan, Meiyan Jiang, Yong Dai, Chih-chen Wang * National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, People’s Republic of China Accepted 14 February 1997
Abstract Human protein disulfide isomerase with an extra 10 amino acid residues of AEITRIDPAM at the N-terminal was expressed in E. coli as a soluble protein comprising 20% of total cell proteins, and was purified to near homogeneity through one step of DEAE-Sephacel chromatography. The mutant enzyme, which had the same CD spectrum and comparable disulfide isomerase and thiol-protein oxidoreductase activities with that of the wild type human and bovine protein disulfide isomerases, also showed chaperone-like activity in stimulating the refolding of proteins containing no disulfide bond. The overall yield of the active product is about 20 mg l − 1 culture. © 1997 Elsevier Science B.V. Keywords: Disulfide isomerase; Protein refolding; Chaperone; High-level expression; Purification
1. Introduction It is now generally accepted that the folding and assembly of nascent peptides into functional proteins in most cases require the assistance of other proteins, which can be classified as molecular chaperones and foldases (Gething and Sambrook, 1992). Protein disulfide isomerase (PDI, EC 5.3.4.1), a multifunctional protein of 57 kDa in the lumen of endoplasmic reticulum and one of * Corresponding author. Tel.: +86 10 62022027; fax: + 86 10 62022026; e-mail: [email protected]
the two foldases so far characterized, is involved in protein folding by catalyzing the formation of native disulfide bonds of nascent peptides (Noiva, 1994). In addition to its isomerase activity, it has been suggested (Wang and Tsou, 1993) and demonstrated recently that PDI has a chaperonelike activity in assisting the reactivation upon dilution of guanidine hydrochloride (GuHCl) denatured proteins containing no disulfide bond (Cai et al., 1994; Song and Wang, 1995). Similar results have been reported for disulfide containing proteins (Puig and Gilbert, 1994). In addition, it has been suggested that the essential function of
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Y. Gao et al. / Journal of Biotechnology 54 (1997) 105–112
yeast PDI does not reside in its isomerase activity as a yeast mutant with PDI inactivated by mutation at the active sites is still viable (LaMantia and Lennarz, 1993). However, little is known on the mechanisms underlying the disulfide isomerase and chaperone activities of PDI. Genes for PDI of rat (Gilbert et al., 1991; Laboissiere et al., 1995), murine (Haugejorden et al., 1992), human (Vuori et al., 1992a; Tojo et al., 1994) and yeast (LaMantia and Lennarz, 1993) have been cloned and expressed in E. coli, yeast or insect cells with a wide range of yields from 2–30 mg PDI l − 1 culture. PDI mutants have also been made for studies on relationship of structure and function (LaMantia and Lennarz, 1993; Vuori et al., 1992b,c; Puig et al., 1994; Lyles and Gilbert, 1994; Darby and Creighton, 1995). PDI has been shown to assist refolding and therefore improve the in vitro renaturation yield of recombinant products expressed as inclusion bodies (Buchner et al., 1992; Tang et al., 1994) and to increase the secretion of foreign proteins in yeast cells when PDI itself is overexpressed (Robinson et al., 1994). In order to elucidate the structural basis of the isomerase and chaperone activities of PDI and mechanism of its function, it is desirable to construct a system for expression of PDI with high yield and a simple purification procedure. In this paper we reported the expression of wild type human PDI (rhPDI) and a mutant human PDI with extra 10 amino acid residues at the N-terminal (mhPDI) as soluble cytoplasmic protein in E. coli. The mhPDI was expressed with much higher yield than that for rhPDI without losing any activities of the wild type enzyme, such as disulfide isomerase, thiol-protein oxidoreductase (TPOR) and chaperone activities.
2. Materials and methods
2.1. Construction of expression 6ector pBV-rhPDI and pBV-mhPDI The plasmid pBR322-PDI, containing the full length human PDI cDNA, was generously pro-
vided by Prof K. Kivirikko (Pihlajaniemi et al., 1987). The 5% noncoding region of PDI gene was removed and then subcloned into pBluescript II SK(-) phagemid (Stratagene) to generate the plasmid BS-PDI (Gao and Wang, 1996). The construction of the expression vector pBV-rhPDI is shown in Fig. 1A. The EcoRI/BamHI-digested 1.7-kb pairs fragment from the plasmid, BS-PDI, containing the coding frame of the hPDI was cloned into the expression vector, pBV220 (Zhang et al., 1990). BS%-PDI was derived from the BS-PDI plasmid but with the insert of human PDI cDNA in a reversed direction. The DNA fragment I of 53 bp was synthesized on an Applied Biosystems DNA Synthesizer 381A (Fig. 1B). The DNA fragment II of about 0.4 kb pairs was generated from BS%-PDI with A6aI, mung bean nuclease and PstI sequential digestion. The DNA fragment III was obtained from BS%-PDI after PstI and SalI digestion. For expression of mhPDI, the pBV-mhPDI was constructed with above three DNA fragments and the digested pBV220 with EcoRI, mung bean nuclease and SalI (Fig. 1C).
2.2. Purification of bPDI, mhPDI and rhPDI The bPDI was prepared from bovine liver essentially according to Lambert and Freedman (1983). E. coli DH5a cells bearing pBV-mhPDI were grown for 4–5 h after induction at 42°C and harvested by centrifugation. The cell pellet was resuspended in lysis buffer (0.1 M Tris, pH 8.0, 0.01% lysozyme) and incubated at 30°C for 2–3 h. Ammonium sulfate was added to the supernatant of the lysate to 85% saturation and the precipitate was dissolved in 10 mM phosphate buffer, pH 6.3. After thorough dialysis, it was then purified by DEAE-Sephacel chromatography in the same buffer with a linear gradient of 0.1–0.6 M NaCl. The mhPDI fractions were identified by isomerase activity assay and pooled at 0.4–0.5 M NaCl (Fig. 2A), desalted and lyophilized. The simple procedure for purifying mhPDI is not enough for the purification of rhPDI expressed in E. coli DH5a probably because of its low level expression and the fact that additional chromatography with hy-
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107
Fig. 1. (A) Construction of the expression vector pBV-rhPDI. (B) The 5% regions and the corresponding amino acid residues at the N-terminal of products of the wild type and the mutant human PDI genes. (C) Construction of the vector pBV-mhPDI for high-level expression of mhPDI.
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Fig. 1. (Cont.)
droxylapatite column and Mono-Q FPLC are employed following DEAE-Sephacel chromatography.
Chaperone-like activity was examined according to Cai et al. (1994).
2.4. CD spectrum determinations 2.3. Acti6ity assay The disulfide isomerase and TPOR activities of wild and mutant PDI preparations were assayed according to Lambert and Freedman (1983).
CD spectrum determinations in the far ultraviolet region from 200–250 nm were carried out with a Jasco J-720 spectropolarimeter.
Y. Gao et al. / Journal of Biotechnology 54 (1997) 105–112
3. Results
Table 1 Enzymatic activities of mhPDI, rhPDI and bPDI
3.1. High-le6el expression in E. coli and purification of mhPDI The rhPDI was expressed with vector pBV220
109
Isomerase (U g−1) TPOR (U g−1)
mhPDI
rhPDI
bPDI
1290 248
930 220
890 230
containing a PRPL promoter in E. coli as a cytoplasmic protein with an unsatisfactory yield. In order to improve the expression level the human PDI cDNA was extended at its 5% region by adding 39 bases. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins of E. coli DH5a cells bearing the mhPDI cDNA as above showed that the mhPDI was expressed as a cytosolic protein constituting about 20% of total cell proteins and 40% of total proteins in the lysate supernatant (Fig. 2B). The preparation of mhPDI with higher than 90% purity was obtained from the supernatant of the cell lysate by ammonium sulfate precipitation and a single step of DEAESephacel chromatography (Fig. 2) with a final yield of about 20 mg l − 1 culture. The final rhPDI preparation is 95% pure.
3.2. Enzyme acti6ities and secondary structure of mhPDI
Fig. 2. Characterization and purification of expressed mhPDI. (A) DEAE-Sephacel chromatography of the lysate supernatant was carried out with a linear gradient of 0.1–0.6 M NaCl in 10 mM phosphate buffer, pH 6.3. The arrow indicates the mhPDI peak. (B) SDS-PAGE was performed using Bio-Rad Mini-Gel system with 10% gel. 1 and 6, Pharmacia HMW marker; 2, total Iysate proteins of DH5a with pBV220; 3, total cell proteins of DH5a bearing pBV-mhPDI; 4, total proteins of lysate supernatant; 5, purified mhPDI after DEAE-Sephacel chromatography.
PDI catalyzes the oxidation, reduction and isomerization of protein disulfides depending on the redox potentials in the reaction medium (Lambert and Freedman, 1983). Ribonuclease with scrambled disulfides and insulin were commonly used as substrates for determinations of isomerase and TPOR activities, respectively (Lambert and Freedman, 1983). Table 1 shows that the isomerase and TPOR activities of purified mhPDI are both in the normal ranges of activities of rhPDI and bPDI preparations. As shown in Fig. 3, the circular dichronism spectrum of mhPDI is almost identical to that of rhPDI and bPDI, suggesting that the N-terminal lengthened enzyme has secondary structures closely similar to that of the native enzyme. The addition of 10 amino acid residues AEITRIDPAM at the N-terminal has no significant effect on either the secondary structure or the enzyme activities.
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3.3. Chaperone-like acti6ity of mhPDI It has been widely accepted that molecular chaperones assist protein folding through binding with folding intermediates to prevent their incorrect interactions resulting in aggregation, so as to favor the productive folding pathway and therefore increase the yield of the native protein. In the system established in this laboratory the reactivation and suppression of aggregation of GuHCl denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) upon dilution in the presence and absence of various PDI preparations were used to measure the chaperone activity of PDI preparations (Cai et al., 1994). Fig. 4A shows that the reactivation of GAPDH increases from 4–18% when the molar ratio of mhPDI to GAPDH (in promoter) is increased from 0–6 with no further increase at higher mhPDI concentrations, which is closely similar to the effect of rhPDI and bPDI on the reactivation of GAPDH. In contrast, bovine serum albumin at a molar ratio of 5 has no effect on the reactivation yield of denatured GAPDH. At the same time the severe aggregation of GAPDH during spontaneous refolding monitored by light scattering was suppressed with increasing
Fig. 4. Chaperone-like activity of PDI preparations. (A) Effect of concentrations of PDI preparations on the reactivation of GuHCl denatured GAPDH at 2.8 mM. Concentrations of rhPDI (), mhPDI () and bPDI ( ) were as indicated. Bovine serum albumin of 14 mM (") was used as control. (B) Effect of concentrations of mhPDI on the aggregation of denatured GAPDH upon dilution as monitored by light scattering. The numbers indicate the molar ratios of mhPDI to GAPDH in promoter.
Fig. 3. The CD spectra of PDI preparations. Protein concentrations were 5.53, 3.96 and 3.74 mM for (—) mhPDI; (- -) bPDI and (···) rhPDI, respectively. Cuvettes of l mm pathlength were used for CD measurements.
concentrations of mhPDI present in the refolding system as shown in Fig. 4B, which is also very similar to the effects of bPDI on the aggregation of GAPDH (Cai et al., 1994). All the above indicate that mhPDI, as well as rhPDI and bPDI, showed chaperone-like activity to suppress aggregation of GAPDH during refolding and thereby assist its correct folding and increase the reactivation yield.
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4. Discussion
Acknowledgements
PDI catalyzes the formation and rearrangement of disulfide bonds of proteins and plays a critical role in nascent peptide folding. In addition the multifunctional PDI displays molecular chaperone activity. However, it is less sure if the enzyme plays different roles either under different conditions or simultaneously. The addition of 30 bp following the ATG start codon at the 5% terminal of the hPDI cDNA with favorable codons of prokaryotic genes designed to enhance translation initiation (Grosjean and Fiers, 1982) does increase the expression level of mhPDI to 20% of the total cell proteins and 40% of the total proteins in the lysate supernatant in contrast to the expression of rhPDI of only a few percent. The sequence between the Shine-Dalgarno region and the start codon has also been extended and changed to A + T rich bases, which may function to increase translation efficiency (Gold et al., 1981). The present study provides a system adequate for the preparation of large amounts of mhPDI with higher than 90% homogeneity from the cell lysate through a simple procedure, ammonium sulfate precipitation followed by one step of DEAE-Sephacel chromatography. The product retains the same enzymatic and chaperone activities as that of the wild type human and bovine PDI, the purification of which often requires a lengthy procedure to give a similar purity. The addition of 10 amino acid residues at the N-terminal has no negative effect on the activities of PDI. The mutant protein appears to be even slightly more active in both the isomerase and the oxidoreductase activities suggesting that the extra stretch of 10 amino acid residues at the N-terminus does not interfere with the structure of either the active sites or the peptide binding sites, as peptide binding site is necessary for both isomerase activity and chaperone activity (Quan et al., 1995; Hayano et al., 1995). Actually the secondary structure of the PDI molecule was also not affected by the N-terminal extension of 10 amino acid residues as shown by CD measurement. Therefore, the mutant PDI can be taken as native PDI in basic studies and applied in protein biotechnology.
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