High-level production of human collagen prolyl 4-hydroxylase in Escherichia coli

doi:10.1016/j.matbio.2004.11.004

Matrix Biology

Volume 24, Issue 1, February 2005, Pages 59-68

https://doi.org/10.1016/j.matbio.2004.11.004 Get rights and content

Abstract

The collagen prolyl 4-hydroxylases (C-P4Hs), enzymes residing within the lumen of the endoplasmic reticulum, play a central role in the synthesis of all collagens. The vertebrate enzymes are α₂β₂ tetramers in which the two catalytic sites are located in the α subunits, and protein disulfide isomerase serves as the β subunit. All attempts to assemble an active C-P4H tetramer from its subunits in in vitro cell-free systems have been unsuccessful, but assembly of a recombinant enzyme has been reported in several cell types by coexpression of the two types of subunit. An active type I C-P4H tetramer was obtained here by periplasmic expression in Escherichia coli strains BL21 and RB791. Further optimization for production by stepwise regulated coexpression of its subunits in the cytoplasm of a thioredoxin reductase and glutathione reductase mutant E. coli strain resulted in large amounts of human type I C-P4H tetramer. The specific activity of the C-P4H tetramer purified from the cytoplasmic expression was within the range of values reported for human type I C-P4H isolated as a nonrecombinant enzyme or produced in the endoplasmic reticulum of insect cells, but the expression level, about 25 mg/l in a fermenter, is about 5–10 times that obtained in insect cells. The enzyme expressed in E. coli differed from those present in vivo and those produced in other hosts in that it lacked the N glycosylation of its α subunits, which may be advantageous in crystallization experiments.

Introduction

The collagen prolyl 4-hydroxylases (C-P4Hs, EC 1.14.11.2), enzymes residing within the lumen of the endoplasmic reticulum (ER), catalyze the formation of 4-hydroxyproline in collagens and more than 20 other proteins with collagen-like sequences. The hydroxylation of proline residues in the Y positions of the collagenous -Gly-X-Y- sequences is essential for the generation of stable collagen triple helices at physiological temperatures (Myllyharju and Kivirikko, 2004). The vertebrate C-P4Hs are α₂β₂ tetramers in which the two catalytic sites are located in the α subunits and the β subunit is identical to the multifunctional enzyme and chaperone protein disulfide isomerase (PDI; Kivirikko and Myllyharju, 1998, Kivirikko and Pihlajaniemi, 1998, Myllyharju, 2003). Three α subunit isoforms characterized in vertebrates have been shown to form [α(I)]₂β₂, [α(II)]₂β₂ and [α(III)]₂β₂ tetramers called type I, II and III C-P4Hs, respectively (Helaakoski et al., 1989, Helaakoski et al., 1995, Annunen et al., 1997, Kukkola et al., 2003, Van Den Diepstraten et al., 2003). The type I enzyme is the major form in most cell types and tissues, but type II is the main form in chondrocytes and endothelial cells, while type III is expressed in several tissues, but at much lower levels (Annunen et al., 1998, Nissi et al., 2001, Kukkola et al., 2003, Van Den Diepstraten et al., 2003).

All attempts to assemble an active C-P4H tetramer from its subunits in in vitro cell-free systems have been unsuccessful (Kivirikko and Myllyharju, 1998, Kivirikko and Pihlajaniemi, 1998), but assembly of an active recombinant human C-P4H tetramer has been reported in various cell types by coexpression of the two types of subunit (Vuori et al., 1992a, Vuorela et al., 1997, John et al., 1999, Toman et al., 2000, Merle et al., 2002). This has enabled detailed studies to be made of the catalytic properties of the recombinant enzyme (Lamberg et al., 1995, Myllyharju and Kivirikko, 1997) and identification and characterization of its peptide substrate-binding domain (Myllyharju and Kivirikko, 1999, Hieta et al., 2003, Pekkala et al., 2004).

Site-directed mutagenesis has shown that two intrachain disulfide bonds in the catalytic α subunit are essential for assembly of the C-P4H tetramer (John and Bulleid, 1994, Lamberg et al., 1995), whereas the functioning of PDI as the β subunit is not dependent on its disulfide isomerase activity, as a mutant PDI polypeptide in which both -Cys-Gly-His-Cys- catalytic sites have been inactivated by mutation to -Ser-Gly-His-Cys- forms a fully active enzyme tetramer (Vuori et al., 1992b). The main function of PDI in the C-P4H tetramer is to keep the highly insoluble α subunits in a catalytically active, nonaggregated conformation (Kivirikko and Myllyharju, 1998, Kivirikko and Pihlajaniemi, 1998, Myllyharju, 2003). PDI has a similar role in the microsomal triglyceride transfer protein dimer, so that this property may be related to its chaperone function (Wetterau et al., 1990, Wetterau et al., 1991).

Many recombinant proteins have been shown to fold correctly in the periplasm of Escherichia coli (Bessette et al., 1999, Swartz, 2001). Novel mutant E. coli strains with a more oxidizing cytoplasm have recently been developed, and model proteins with up to 17 disulfide bonds have now been successfully expressed in such strains (Bessette et al., 1999). We report here that large amounts of a fully active recombinant human C-P4H tetramer can be assembled in the cytoplasm of a thioredoxin reductase and glutathione reductase double mutant E. coli strain by coexpression of the two types of subunit. Cytoplasmic expression resulted in higher amounts of active tetramer than were obtained in periplasmic expression. Coexpression of the chaperone immunoglobulin heavy chain binding protein (BiP), which is known to form soluble α subunit–BiP complexes as intermediates in the synthesis of the enzyme tetramer within the ER of mammalian cells (John and Bulleid, 1996), did not increase the amount of C-P4H tetramer in the E. coli periplasmic or cytoplasmic fractions.

Section snippets

Expression of recombinant human type I C-P4H in the periplasm of E. coli

The C-P4H tetramer is a soluble ER luminal protein, the two intrachain disulfide bonds in the catalytic α subunit being essential for its assembly (John and Bulleid, 1994, Lamberg et al., 1995). Recombinant expression of active human type I C-P4H in E. coli was obtained using a plasmid encoding the α(I) and PDI polypeptides possessing periplasmic signal sequences. A cDNA encoding the human α(I) subunit was cloned into the vector pASK-IBA2 under control from the tetA promoter in-frame with the

Conclusions

Production of an active recombinant human C-P4H tetramer has previously been reported in various cell types (Vuori et al., 1992a, Vuorela et al., 1997, John et al., 1999, Toman et al., 2000, Merle et al., 2002). The present study shows that large amounts of a fully active recombinant human C-P4H tetramer can be assembled in the cytoplasm of E. coli using a specific host strain with a less reducing cytoplasmic environment. Assembly was found to occur without coexpression of recombinant BiP, a

Generation of E. coli expression vectors

To generate an E. coli construct for cytoplasmic expression of human type I C-P4H (Fig. 1A), an oligonucleotide containing EcoRI, SalI and BamHI sites was first inserted into the BstBI-HindIII-digested vector pASK-IBA3 (IBA GmbH). The human α(I) subunit cDNA without its signal sequence but with an additional stop codon TGA and flanked by BsaI sites was generated by PCR using the α(I) cDNA PA59 (Helaakoski et al., 1989) as a template and cloned into the BsaI-digested modified pASK-IBA3 vector

Acknowledgements

The expert technical assistance of Minna Siurua is gratefully acknowledged. We thank Rami Kuivila for assistance with the fermentation experiments. We are also grateful to J. Buchner and J. Veijola for the gifts of plasmids. This study was supported by grants from the Health Science Council (200471) and the Finnish Centre of Excellence Programme 2000–2005 (44843) of the Academy of Finland.

References (34)

P. Annunen et al.
Cloning of the human prolyl 4-hydroxylase α subunit isoform α(II) and characterization of the type II enzyme tetramer. The α(I) and α(II) subunits do not form a mixed α(I)α(II)β₂ tetramer
J. Biol. Chem.
(1997)
P. Annunen et al.
The novel type II prolyl 4-hydroxylase is the main enzyme form in chondrocytes and capillary endothelial cells, whereas the type I enzyme predominates in most cells
J. Biol. Chem.
(1998)
R. Hieta et al.
The peptide-substrate binding domain of human collagen prolyl 4-hydroxylases. Backbone assignments, secondary structure and binding of proline-rich peptides
J. Biol. Chem.
(2003)
K.I. Kivirikko et al.
Prolyl 4-hydroxylases and their protein disulfide isomerase subunit
Matrix Biol.
(1998)
K.I. Kivirikko et al.
Posttranslational enzymes in the biosynthesis of collagen: intracellular enzymes
Methods Enzymol.
(1982)
K.I. Kivirikko et al.
Recent developments in posttranslational modification: intracellular processing
Methods Enzymol.
(1987)
G. Knarr et al.
BiP-binding sequences in HIV gp160. Implications for the binding specificity of BiP
J. Biol. Chem.
(1999)
L. Kukkola et al.
Identification and characterization of a third human, rat and mouse collagen prolyl 4-hydroxylase isoenzyme
J. Biol. Chem.
(2003)
A. Lamberg et al.
Site-directed mutagenesis of the alpha subunit of human prolyl 4-hydroxylase. Identification of three histidine residues critical for catalytic activity
J. Biol. Chem.
(1995)
C. Merle et al.
Hydroxylated human homotrimeric collagen I in Agrobacterium tumefaciens-mediated transient expression and in transgenic tobacco plant
FEBS Lett.
(2002)

J. Myllyharju

Prolyl 4-hydroxylases, the key enzymes of collagen biosynthesis

Matrix Biol.

(2003)

M. Pekkala et al.

The peptide-substrate-binding domain of collagen prolyl 4-hydroxylases is a TPR domain with functional aromatic residues

J. Biol. Chem.

(2004)

J.R. Swartz

Advances in Escherichia coli production of therapeutic proteins

Curr. Opin. Biotechnol.

(2001)

P.D. Toman et al.

Production of recombinant human type I procollagen trimers using a four-gene expression system in the yeast Saccharomyces cerevisiae

J. Biol. Chem.

(2000)

K. Vuori et al.

Expression and site-directed mutagenesis of human protein disulfide isomerase in Escherichia coli

J. Biol. Chem.

(1992)

J.R. Wetterau et al.

Protein disulfide isomerase is a component of the microsomal triglyceride transfer protein complex

J. Biol. Chem.

(1990)

P.H. Bessette et al.

Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm

Proc. Natl. Acad. Sci. U. S. A.

(1999)

Cited by (28)

Co-expression of recombinant human collagen α1(III) chain with viral prolyl 4-hydroxylase in Pichia pastoris GS115
2023, Protein Expression and Purification
Citation Excerpt :
Recently, some nonhuman P4Hs were considered. Several researchers have shown that the co-expression of the P4H α and PDI/β genes in Escherichia coli (E.coli) could produce active P4H [14]. In addition, some plant-derived P4H exhibited substrate preference [15].
The Collagen α1(Ш) chain (COL3A1) is an important structural protein on the surface of human skin. The activity of prolyl 4-hydroxylase (P4H) is crucial to maintaining the stable triple-helix structure and function of human COL3A1. To obtain hydroxylated human COL3A1, virus-derived P4H A085R was co-expressed with human COL3A1 in Pichia pastoris GS115. Colony PCR analysis and sequencing after transfection confirmed that the target gene was successfully inserted. Quantitative reverse transcription PCR (RT-qPCR) indicated that human COL3A1 and P4H A085R were expressed at mRNA levels in the clones. SDS-PAGE and Western blot analysis of supernatant from the recombinant methylotrophic yeast culture showed that recombinant human COL3A1 (rhCOL3A1) was secreted into the culture medium with an apparent molecular mass of approximately 130 kDa. It was observed that the amount of secreted rhCOL3A1 was highest at 120 h after induction. Furthermore, mass spectrometry analysis demonstrated that rhCOL3A1 was successfully expressed in P. pastoris. The His-tagged rhCOL3A1 protein was purified by Ni-affinity column chromatography.
Production of disulfide-bonded proteins in Escherichia coli
2012, Protein Expression and Purification
Citation Excerpt :
These differences between the two compartments can have a significant effect on the final yields of a protein. For example, the yield of collagen prolyl 4-hydrolase was ∼20 times higher in the cytoplasm of FÅ113 (Origami) than in the periplasm of the corresponding BL21 wild type strain and ∼10 times better than when expressed in insect cells [146]. Our current view on the mechanism of disulfide bond formation in the cytoplasm of FÅ113 is incomplete.
Disulfide bonds are covalent bonds formed post-translationally by the oxidation of a pair of cysteines. A disulfide bond can serve structural, catalytic, and signaling roles. However, there is an inherent problem to the process of disulfide bond formation: mis-pairing of cysteines can cause misfolding, aggregation and ultimately result in low yields during protein production. Recent developments in the understanding of the mechanisms involved in the formation of disulfide bonds have allowed the research community to engineer and develop methods to produce multi-disulfide-bonded proteins to high yields. This review attempts to highlight the mechanisms responsible for disulfide bond formation in Escherichia coli, both in its native periplasmic compartment in wild-type strains and in the genetically modified cytoplasm of engineered strains. The purpose of this review is to familiarize the researcher with the biological principles involved in the formation of disulfide-bonded proteins with the hope of guiding the scientist in choosing the optimum expression system.
Optimization of Fermentation Process for Human-like Collagen Production of Recombinant Escherichia coli Using Response Surface Methodology
2010, Chinese Journal of Chemical Engineering
In order to improve the production of human-like collagen III (HLC III) by fed-batch culture of recombinant Escherichia coli BL21, the Plackett-Burman and Box-Behnken design were applied to optimize the fermentation process parameters. Three variables (induction time, inoculum age and pH), which have significant effects on HLC III production, were selected from eight variables by Plackett-Burman design. With the regression coefficient analysis in the Box-Behnken design, a relationship between HLC III production and three significant factors was obtained, and the optimum levels of the three variables were as follows: induction time 3.2h, inoculum age 12.6 h and pH 6.7. The 3D response surface plots and 2D contour plots created by the Box-Behnken design showed that the interaction between induction time and pH and that between innoculum age and pH were significant. An average 9.68 g·L⁻¹HLC III production was attained in the validation experiment under optimized condition, which was 80% higher than the yield of 5.36 g·L⁻¹ before optimization.
Direct and continuous assay for prolyl 4-hydroxylase
2009, Analytical Biochemistry
Prolyl 4-hydroxylase (P4H) is a nonheme iron dioxygenase that catalyzes the posttranslational hydroxylation of (2S)-proline (Pro) residues in protocollagen strands. The resulting (2S,4R)-4-hydroxyproline (Hyp) residues are essential for the folding, secretion, and stability of the collagen triple helix. P4H uses α-ketoglutarate and O₂ as cosubstrates, and forms succinate and CO₂ as well as Hyp. Described herein is the first assay for P4H that continuously and directly detects turnover of the proline-containing substrate. This assay is based on (2S,4S)-4-fluoroproline (flp), a proline analogue that is transformed into (2S)-4-ketoproline (Kep) and inorganic fluoride by P4H. The fluoride ion, and thus turnover by P4H, is detected by a fluoride ion-selective electrode. Using this assay, steady-state kinetic parameters for the human P4H-catalyzed turnover of a flp-containing peptide were determined and found to be comparable to those obtained with a discontinuous HPLC-based assay. In addition, this assay can be used to characterize P4H variants, as demonstrated by a comparison of catalysis by D414A P4H and the wild-type enzyme. Finally, the use of the assay to identify small-molecule inhibitors of P4H was verified by an analysis of catalysis in the presence of 2,4-pyridine dicarboxylate, an analogue of α-ketoglutarate. Thus, the assay described herein could facilitate biochemical analyses of this essential enzyme.
Recombinant human SMOCs produced by in vitro refolding: Calcium-binding properties and interactions with serum proteins
2008, Protein Expression and Purification
Secreted modular calcium-binding (SMOC) proteins are little known members of the BM-40 family of matricellular proteins. SMOC-1 is localized in basement membranes, while SMOC-2 exhibits pro-angiogenic properties and stimulates cell cycle progression via integrin-linked kinase. In this work we have expressed recombinant human SMOCs in inclusion bodies in Escherichia coli. Soluble proteins were prepared by in vitro refolding with a final yield of approximately 3 mg of purified SMOCs per liter of bacterial culture. The folding state of the products and their ability to reversibly bind calcium ions were verified by CD spectroscopy. The EF hands of the refolded SMOCs were both functional, one had high affinity for calcium ions (K_d values in the 0.7–1 μM range), while the other had lower affinity (K_d values 20–25 μM). The proteins were also examined for their ability to bind blood serum proteins. Three of the bands specifically retained on SMOC-Sepharose were identified as C-reactive protein, an acute phase protein from the pentraxin family, the basement membrane and elastic fiber-associated fibulin-1, and vitronectin, which is involved in cell adhesion, migration and proliferation and binds numerous extracellular and membrane proteins, including integrins. The interactions were additionally confirmed in solution using purified individual proteins by the method of biotin label transfer from one interacting partner to the other. Their identification is among the first pieces of information about the action of the SMOCs on molecular level and opens new possibilities for future research aimed towards elucidating the physiological roles of these versatile proteins.
Fermentation process for tetrameric human collagen prolyl 4-hydroxylase in Escherichia coli: Improvement by gene optimisation of the PDI/β subunit and repeated addition of the inducer anhydrotetracycline
2007, Journal of Biotechnology
The collagen prolyl 4-hydroxylases (C-P4Hs) that reside within the lumen of the endoplasmic reticulum (ER) are the key enzymes in the biosynthesis of collagens. The vertebrate enzymes are α₂β₂ tetramers consisting of two catalytic α subunits and two β subunits that are identical to protein disulfide isomerase (PDI). Cytoplasmic production of an active human C-P4H has recently been described in the Origami (trxB gor) mutant Escherichia coli using a bicistronic vector with independent control of the α and PDI/β subunit expression by the tetA and T5-lac promoters, respectively, enabling sequential induction (Neubauer, A., Neubauer, P., Myllyharju, J., 2005. High-level production of human collagen prolyl 4-hydroxylase in Escherichia coli. Matrix Biol. 24, 59–68). We show here that the yield of active C-P4H in shake flasks is increased 50-fold by improving the expression level of the PDI/β subunit through gene optimisation. We also found that stable expression of the α subunit mRNA in a fed-batch fermentation process requires repeated additions of anhydrotetracycline. This finding may be of a wider general importance for the use of the tetA promoter in fed-batch cultivations, especially if recombinant proteins are expressed during long production phases. We also show that growth of the E. coli Origami strain to high cell density on a complex medium with consecutive sequential induction is difficult to achieve and that optimisation of similarly complicated systems can greatly benefit from the use of quantitative mRNA analysis for the evaluation of transcriptional bottlenecks. The optimisation approach resulted in a fermentation yield of 143 mg L⁻¹ of active C-P4H, corresponding to approximately 7.5% of the total soluble cell protein.

View all citing articles on Scopus

View full text

High-level production of human collagen prolyl 4-hydroxylase in Escherichia coli

Abstract

Introduction

Section snippets

Expression of recombinant human type I C-P4H in the periplasm of E. coli

Conclusions

Generation of E. coli expression vectors

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Matrix Biol.

Methods Enzymol.

Methods Enzymol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

Matrix Biol.

J. Biol. Chem.

Curr. Opin. Biotechnol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm

Proc. Natl. Acad. Sci. U. S. A.