Assembly of Stable Human Type I and III Collagen Molecules from Hydroxylated Recombinant Chains in the Yeast Pichia pastoris

The C-propeptides of the proα chains of type I and type III procollagens are believed to be essential for correct chain recognition and chain assembly in these molecules. We studied here whether the 30-kDa C-propeptides of the human pCα1(I), pCα2(I), and pCα1(III) chains, i.e. proα chains lacking their N-propeptides, can be replaced by foldon, a 29-amino acid sequence normally located at the C terminus of the polypeptide chains in the bacteriophage T4 fibritin. The αfoldon chains were expressed in Pichia pastoris cells that also expressed the two types of subunit of human prolyl 4-hydroxylase; the foldon domain was subsequently removed by pepsin treatment, which also digests non-triple helical collagen chains, whereas triple helical collagen molecules are resistant to it. The foldon domain was found to be very effective in chain assembly, as expression of the α1(I)foldon or α1(III)foldon chains gave about 2.5–3-fold the amount of pepsin-resistant type I or type III collagen homotrimers relative to those obtained using the authentic C-propeptides. In contrast, expression of chains with no oligomerization domain led to very low levels of pepsin-resistant molecules. Expression of α2(I)foldon chains gave no pepsin-resistant molecules at all, indicating that in addition to control at the level of the C-propeptide other restrictions at the level of the collagen domain exist that prevent the formation of stable [α2(I)]3 molecules. Co-expression of α1(I)foldon and α2(I)foldon chains led to an efficient assembly of heterotrimeric molecules, their amounts being about 2-fold those obtained with the authentic C-propeptides and the α1(I) to α2(I) ratio being 1.91 ± 0.31 (S.D.). As the foldon sequence contains no information for chain recognition, our data indicate that chain assembly is influenced not only by the C-terminal oligomerization domain but also by determinants present in the α chain domains. The C-propeptides of the proα chains of type I and type III procollagens are believed to be essential for correct chain recognition and chain assembly in these molecules. We studied here whether the 30-kDa C-propeptides of the human pCα1(I), pCα2(I), and pCα1(III) chains, i.e. proα chains lacking their N-propeptides, can be replaced by foldon, a 29-amino acid sequence normally located at the C terminus of the polypeptide chains in the bacteriophage T4 fibritin. The αfoldon chains were expressed in Pichia pastoris cells that also expressed the two types of subunit of human prolyl 4-hydroxylase; the foldon domain was subsequently removed by pepsin treatment, which also digests non-triple helical collagen chains, whereas triple helical collagen molecules are resistant to it. The foldon domain was found to be very effective in chain assembly, as expression of the α1(I)foldon or α1(III)foldon chains gave about 2.5–3-fold the amount of pepsin-resistant type I or type III collagen homotrimers relative to those obtained using the authentic C-propeptides. In contrast, expression of chains with no oligomerization domain led to very low levels of pepsin-resistant molecules. Expression of α2(I)foldon chains gave no pepsin-resistant molecules at all, indicating that in addition to control at the level of the C-propeptide other restrictions at the level of the collagen domain exist that prevent the formation of stable [α2(I)]3 molecules. Co-expression of α1(I)foldon and α2(I)foldon chains led to an efficient assembly of heterotrimeric molecules, their amounts being about 2-fold those obtained with the authentic C-propeptides and the α1(I) to α2(I) ratio being 1.91 ± 0.31 (S.D.). As the foldon sequence contains no information for chain recognition, our data indicate that chain assembly is influenced not only by the C-terminal oligomerization domain but also by determinants present in the α chain domains. The collagen superfamily of proteins includes more than 20 types of collagen and more than 15 additional proteins that have collagen-like domains. All collagen molecules consist of three polypeptide chains, called α chains, 1The abbreviations used are: α chain, a collagen polypeptide chain; proα chain, a procollagen polypeptide chain; pCα chain, a procollagen polypeptide lacking the N-propeptide; αfoldon chain, a pCα chain with the C-propeptide replaced by the foldon domain.1The abbreviations used are: α chain, a collagen polypeptide chain; proα chain, a procollagen polypeptide chain; pCα chain, a procollagen polypeptide lacking the N-propeptide; αfoldon chain, a pCα chain with the C-propeptide replaced by the foldon domain. that are coiled around each other into a triple helix and contain the triplet sequence -Gly-X-Y-, in which the Y position amino acid is often 4-hydroxyproline. The most abundant collagens form fibrils and are therefore known as fibril-forming collagens, whereas others form other kinds of supramolecular structures. The molecules of the most abundant fibril-forming collagen, type I, consist of two α1(I) chains and one α2(I) chain, whereas the molecules of type III collagen are [α1(III)]3 homotrimers. In addition to the type I collagen heterotrimer, most tissues also contain a small amount of type I collagen with a chain composition of [α1(I)]3, known as the type I collagen homotrimer (for reviews, see Refs. 1Kadler K. Protein Profile. 1995; 2: 491-619PubMed Google Scholar, 2Prockop D.J. Kivirikko K.I. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1355) Google Scholar, 3Bateman J.F. Lamandé S.R. Ramshaw J.A.M. Comper W.D. Extracellular Matrix. Vol. 2. Harwood, Amsterdam1996: 22-67Google Scholar, 4Myllyharju J. Kivirikko K.I. Ann. Med. 2001; 33: 7-21Crossref PubMed Scopus (534) Google Scholar). The fibril-forming collagens are synthesized as procollagen molecules with N- and C-terminal propeptides. Chain assembly begins with association of the three C-propeptides through a process directed by their structures (2Prockop D.J. Kivirikko K.I. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1355) Google Scholar, 5McLaughlin S.H. Bulleid N.J. Matrix Biol. 1998; 16: 369-377Crossref PubMed Scopus (97) Google Scholar). Renaturation experiments with individual type I collagen α chains that lack any propeptides have indicated, however, that the chains form both [α(I)]2α2(I) heterotrimers and [α1(I)]3 homotrimers, although the process is very slow and the T m values of the molecules formed are lower than of those present in vivo (6Tkocz C. Kühn K. Eur. J. Biochem. 1969; 7: 454-462Crossref PubMed Scopus (87) Google Scholar, 7Leikina E. Mertts M.V. Leikin S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1314-1318Crossref PubMed Scopus (439) Google Scholar). Based on these findings and numerous subsequent studies carried out in a large variety of biological systems over a period of more than 25 years, the C-propeptides are now believed to be essential for correct chain recognition and to play a crucial role in chain assembly in vivo (2Prockop D.J. Kivirikko K.I. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1355) Google Scholar, 5McLaughlin S.H. Bulleid N.J. Matrix Biol. 1998; 16: 369-377Crossref PubMed Scopus (97) Google Scholar, 8Lees J.F. Tasab M. Bulleid N.J. EMBO J. 1997; 16: 908-916Crossref PubMed Scopus (123) Google Scholar). Most studies of collagen synthesis have used vertebrate cells, which usually possess sufficient levels of all the specific cotranslational and posttranslational enzymes needed for collagen processing. In recent years experiments have also been performed using recombinant expression in cultured insect cells (9Lamberg A. Helaakoski T. Myllyharju J. Peltonen S. Notbohm H. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1996; 271: 11988-11995Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 10Myllyharju J. Lamberg A. Notbohm H. Fietzek P.P. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1997; 272: 21824-21830Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 11Nokelainen M. Helaakoski T. Myllyharju J. Notbohm H. Pihlajaniemi T. Fietzek P.P. Kivirikko K.I. Matrix Biol. 1998; 16: 329-338Crossref PubMed Scopus (49) Google Scholar), yeasts (12Vuorela A. Myllyharju J. Nissi R. Pihlajaniemi T. Kivirikko K.I. EMBO J. 1997; 16: 6702-6712Crossref PubMed Scopus (128) Google Scholar, 13Toman P.D. Chisholm G. McMullin H. Giere L.M. Olsen D.R. Kovach R.J. Leigh S.D. Fong B.E. Chang R. Daniels G.A. Berg R.A. Hitzeman R.A. J. Biol. Chem. 2000; 275: 23303-23309Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 14Nokelainen M. Tu H. Vuorela A. Notbohm H. Kivirikko K.I. Myllyharju J. Yeast. 2001; 18: 797-806Crossref PubMed Scopus (85) Google Scholar) and plants (15Merle C. Perret S. Lacour T. Jonval V. Hudaverdian S. Garrone R. Ruggiero F. Theisen M. FEBS Lett. 2002; 27: 114-118Crossref Scopus (87) Google Scholar). These cells have been shown to assemble partially (13Toman P.D. Chisholm G. McMullin H. Giere L.M. Olsen D.R. Kovach R.J. Leigh S.D. Fong B.E. Chang R. Daniels G.A. Berg R.A. Hitzeman R.A. J. Biol. Chem. 2000; 275: 23303-23309Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 15Merle C. Perret S. Lacour T. Jonval V. Hudaverdian S. Garrone R. Ruggiero F. Theisen M. FEBS Lett. 2002; 27: 114-118Crossref Scopus (87) Google Scholar) or fully (9Lamberg A. Helaakoski T. Myllyharju J. Peltonen S. Notbohm H. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1996; 271: 11988-11995Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 10Myllyharju J. Lamberg A. Notbohm H. Fietzek P.P. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1997; 272: 21824-21830Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 11Nokelainen M. Helaakoski T. Myllyharju J. Notbohm H. Pihlajaniemi T. Fietzek P.P. Kivirikko K.I. Matrix Biol. 1998; 16: 329-338Crossref PubMed Scopus (49) Google Scholar, 12Vuorela A. Myllyharju J. Nissi R. Pihlajaniemi T. Kivirikko K.I. EMBO J. 1997; 16: 6702-6712Crossref PubMed Scopus (128) Google Scholar, 14Nokelainen M. Tu H. Vuorela A. Notbohm H. Kivirikko K.I. Myllyharju J. Yeast. 2001; 18: 797-806Crossref PubMed Scopus (85) Google Scholar) hydroxylated recombinant collagen chains into molecules with partially (13Toman P.D. Chisholm G. McMullin H. Giere L.M. Olsen D.R. Kovach R.J. Leigh S.D. Fong B.E. Chang R. Daniels G.A. Berg R.A. Hitzeman R.A. J. Biol. Chem. 2000; 275: 23303-23309Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 15Merle C. Perret S. Lacour T. Jonval V. Hudaverdian S. Garrone R. Ruggiero F. Theisen M. FEBS Lett. 2002; 27: 114-118Crossref Scopus (87) Google Scholar) or fully (9Lamberg A. Helaakoski T. Myllyharju J. Peltonen S. Notbohm H. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1996; 271: 11988-11995Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 10Myllyharju J. Lamberg A. Notbohm H. Fietzek P.P. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1997; 272: 21824-21830Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 11Nokelainen M. Helaakoski T. Myllyharju J. Notbohm H. Pihlajaniemi T. Fietzek P.P. Kivirikko K.I. Matrix Biol. 1998; 16: 329-338Crossref PubMed Scopus (49) Google Scholar, 12Vuorela A. Myllyharju J. Nissi R. Pihlajaniemi T. Kivirikko K.I. EMBO J. 1997; 16: 6702-6712Crossref PubMed Scopus (128) Google Scholar, 14Nokelainen M. Tu H. Vuorela A. Notbohm H. Kivirikko K.I. Myllyharju J. Yeast. 2001; 18: 797-806Crossref PubMed Scopus (85) Google Scholar) stable triple helices, provided that they co-express the two types of subunit of a recombinant prolyl 4-hydroxylase. This key enzyme of collagen synthesis is an α2β2 tetramer in vertebrates (16Kivirikko K.I. Myllyharju J. Matrix Biol. 1998; 16: 357-368Crossref PubMed Scopus (233) Google Scholar, 17Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Relat. Areas Mol. Biol. 1998; 72: 325-400PubMed Google Scholar, 18Myllyharju J. Matrix Biol. 2003; 22: 15-24Crossref PubMed Scopus (317) Google Scholar). Most of the triple helical collagen molecules assembled in insect cells, yeasts, and plants are not secreted (9Lamberg A. Helaakoski T. Myllyharju J. Peltonen S. Notbohm H. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1996; 271: 11988-11995Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 10Myllyharju J. Lamberg A. Notbohm H. Fietzek P.P. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1997; 272: 21824-21830Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 11Nokelainen M. Helaakoski T. Myllyharju J. Notbohm H. Pihlajaniemi T. Fietzek P.P. Kivirikko K.I. Matrix Biol. 1998; 16: 329-338Crossref PubMed Scopus (49) Google Scholar, 12Vuorela A. Myllyharju J. Nissi R. Pihlajaniemi T. Kivirikko K.I. EMBO J. 1997; 16: 6702-6712Crossref PubMed Scopus (128) Google Scholar, 13Toman P.D. Chisholm G. McMullin H. Giere L.M. Olsen D.R. Kovach R.J. Leigh S.D. Fong B.E. Chang R. Daniels G.A. Berg R.A. Hitzeman R.A. J. Biol. Chem. 2000; 275: 23303-23309Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 14Nokelainen M. Tu H. Vuorela A. Notbohm H. Kivirikko K.I. Myllyharju J. Yeast. 2001; 18: 797-806Crossref PubMed Scopus (85) Google Scholar, 15Merle C. Perret S. Lacour T. Jonval V. Hudaverdian S. Garrone R. Ruggiero F. Theisen M. FEBS Lett. 2002; 27: 114-118Crossref Scopus (87) Google Scholar) but accumulate within the endoplasmic reticulum (19Keizer-Gunnink I. Vuorela A. Myllyharju J. Pihlajaniemi T. Kivirikko K.I. Veenhuis M. Matrix Biol. 2000; 19: 29-36Crossref PubMed Scopus (22) Google Scholar). Studies of these systems have confirmed that the N-propeptides are not required for the assembly of triple helical molecules, as they can be swapped between collagen types (10Myllyharju J. Lamberg A. Notbohm H. Fietzek P.P. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1997; 272: 21824-21830Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 11Nokelainen M. Helaakoski T. Myllyharju J. Notbohm H. Pihlajaniemi T. Fietzek P.P. Kivirikko K.I. Matrix Biol. 1998; 16: 329-338Crossref PubMed Scopus (49) Google Scholar) or omitted (14Nokelainen M. Tu H. Vuorela A. Notbohm H. Kivirikko K.I. Myllyharju J. Yeast. 2001; 18: 797-806Crossref PubMed Scopus (85) Google Scholar, 15Merle C. Perret S. Lacour T. Jonval V. Hudaverdian S. Garrone R. Ruggiero F. Theisen M. FEBS Lett. 2002; 27: 114-118Crossref Scopus (87) Google Scholar, 20Olsen D.R. Leigh S.D. Chang R. McMullin H. Ong W. Tai E. Chisholm G. Birk D.E. Berg R.A. Hitzeman R.A. Toman P.D. J. Biol. Chem. 2001; 276: 24038-24043Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). A recent study reported, highly surprisingly, that assembly of triple helical recombinant type I collagen molecules from partially hydroxylated α chains in the yeast Saccharomyces cerevisiae does not even require the C-propeptides (20Olsen D.R. Leigh S.D. Chang R. McMullin H. Ong W. Tai E. Chisholm G. Birk D.E. Berg R.A. Hitzeman R.A. Toman P.D. J. Biol. Chem. 2001; 276: 24038-24043Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The triple helical molecules had an α1(I) to α2(I) chain ratio of 5:1, however, suggesting that they consist of mixtures of [α1(I)]2α2(I) heterotrimers and [α1(I)]3 homotrimers (20Olsen D.R. Leigh S.D. Chang R. McMullin H. Ong W. Tai E. Chisholm G. Birk D.E. Berg R.A. Hitzeman R.A. Toman P.D. J. Biol. Chem. 2001; 276: 24038-24043Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In the present work we have studied whether fully hydroxylated recombinant human type I collagen α chains lacking C-propeptides can assemble effectively into triple helical molecules in the yeast Pichia pastoris when it co-expresses the two types of subunits of a recombinant human prolyl 4-hydroxylase (12Vuorela A. Myllyharju J. Nissi R. Pihlajaniemi T. Kivirikko K.I. EMBO J. 1997; 16: 6702-6712Crossref PubMed Scopus (128) Google Scholar, 14Nokelainen M. Tu H. Vuorela A. Notbohm H. Kivirikko K.I. Myllyharju J. Yeast. 2001; 18: 797-806Crossref PubMed Scopus (85) Google Scholar). As the collagen domains of the α chains of type III collagen, unlike those of type I, contain two cysteine residues at their C-terminal ends, which are involved in the formation of interchain disulfide bonds (see Ref. 21Bulleid N.J. Wilson R. Lees J.F. Biochem. J. 1996; 317: 195-202Crossref PubMed Scopus (62) Google Scholar), we also determined whether type III collagen α chains lacking their C-propeptides would be assembled more effectively than those of type I. One major aspect studied here was whether the 30-kDa C-propeptides of the proα1(I), proα2(I), or proα1(III) chains could be replaced by foldon, a 29-amino acid peptide that is normally located at the C terminus of the polypeptide chains in the bacteriophage T4 fibritin, a three-stranded α helical coiled-coil protein, and appears to be essential for the assembly of the fibritin molecule (22Tao Y. Strelkov S.V. Mesyanzhinov V. Rossmann M.G. Structure. 1997; 5: 789-798Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 23Letarov A.V. Londer Y.Y. Boudko S.P. Mesyanzhinov V.V. Biochemistry (Moscow). 1999; 64: 817-823PubMed Google Scholar, 24Boudko S.P. Londer Y.Y. Letarov A.V. Sernova N.V. Engel J. Mesyanzhinov V.V. Eur. J. Biochem. 2002; 269: 833-841Crossref PubMed Scopus (32) Google Scholar). The addition of foldon to the C terminus of the peptide (Pro-Pro-Gly)10 has been shown to markedly increase the T m of the triple helical 4-hydroxyproline-free molecules that are formed from these peptides even in the absence of foldon (25Frank S. Kammerer R.A. Mechling D. Schulthess T. Landwehr R. Bann J. Guo Y Lustig A. Bächinger H.P. Engel J. J. Mol. Biol. 2001; 308: 1081-1089Crossref PubMed Scopus (161) Google Scholar, 26Boudko S. Frank S. Kammerer R.A. Stetefeld J. Schulthess T. Landwehr R. Lustig A. Bächinger H.P. Engel J. J. Mol. Biol. 2002; 317: 459-470Crossref PubMed Scopus (92) Google Scholar). No data are available to indicate whether foldon could be used to replace the C-propeptides of proα chains in the assembly of stable triple helical collagen molecules. We were unable to confirm the effective assembly of triple helical collagen molecules from α chains lacking the C-propeptides in yeast cells (20Olsen D.R. Leigh S.D. Chang R. McMullin H. Ong W. Tai E. Chisholm G. Birk D.E. Berg R.A. Hitzeman R.A. Toman P.D. J. Biol. Chem. 2001; 276: 24038-24043Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), at least in P. pastoris, but it was clear that the C-propeptides could be replaced by the foldon sequence. Expression of α1(I)foldon or α1(III)foldon chains led to an even more efficient assembly of homotrimeric molecules with stable triple helices than in the cases of chains expressed with their own C-propeptides. Furthermore, co-expression of the α1(I)foldon and α2(I)foldon chains led to the effective formation of type I collagen heterotrimers with the correct 2:1 chain ratio, indicating that the chain composition is determined not only by the C-terminal oligomerization domain but also by determinants present in the collagen domain of the polypeptide chains, at least in the case of very small oligomerization domains such as foldon. P. pastoris Expression Vectors and Generation of Recombinant Strains—The P. pastoris host strain yJC300 (his4, arg4, ade1) and the expression vectors pBLADESX and pBLARGIX (27Cereghino G.P. Cereghino J.L. Sunga A.J. Johnson M.A. Lim M. Gleeson M.A. Cregg J.M. Gene. 2001; 263: 159-169Crossref PubMed Scopus (83) Google Scholar) were gifts from Dr. James Cregg, (Keck Graduate Institute of Applied Life Sciences), and the vectors pPIC3K, pPICZB, and pPICZαA were from Invitrogen. The recombinant strains were generated by the electroporation method according to the manufacturer's instructions (Invitrogen (32InvitrogenManual of Methods for Expression of Recombinant Proteins in Pichia pastoris. Version M, Invitrogen, Carlsbad, CA2002Google Scholar)). The recombinant strains were of the methanol utilization-plus phenotype. PCα1(I), α1(I)foldon, and α1(I) Strains—A cDNA for the β subunit of human prolyl 4-hydroxylase lacking the signal sequence and flanked by EcoRI restriction sites (12Vuorela A. Myllyharju J. Nissi R. Pihlajaniemi T. Kivirikko K.I. EMBO J. 1997; 16: 6702-6712Crossref PubMed Scopus (128) Google Scholar) was cloned into pPICZαA in frame with the S. cerevisiae α mating factor (αMF) pre-pro sequence. The expression cassette encoding the αMF-β polypeptide was digested from pPICZαAβ with BamHI-BglII and cloned into pBLADESX. To generate a recombinant P. pastoris strain expressing human prolyl 4-hydroxylase α2β2 tetramers, the pBLARGIXα (14Nokelainen M. Tu H. Vuorela A. Notbohm H. Kivirikko K.I. Myllyharju J. Yeast. 2001; 18: 797-806Crossref PubMed Scopus (85) Google Scholar) and pBLADESXβ were linearized with HincII and SpeI, respectively, and cotransformed into the yJC300 strain. The resulting strain was named ArgαAdeβ,his–. A P. pastoris expression cassette encoding human type I pCα1 chains (procollagen chains lacking their N-propeptides) was digested from pPICZBpCα1(I) (14Nokelainen M. Tu H. Vuorela A. Notbohm H. Kivirikko K.I. Myllyharju J. Yeast. 2001; 18: 797-806Crossref PubMed Scopus (85) Google Scholar) with PmeI-NotI and cloned into pPIC3K to generate pPIC3KpCα1(I). A cDNA coding for the 29-amino acid foldon domain of fibritin (22Tao Y. Strelkov S.V. Mesyanzhinov V. Rossmann M.G. Structure. 1997; 5: 789-798Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 23Letarov A.V. Londer Y.Y. Boudko S.P. Mesyanzhinov V.V. Biochemistry (Moscow). 1999; 64: 817-823PubMed Google Scholar, 24Boudko S.P. Londer Y.Y. Letarov A.V. Sernova N.V. Engel J. Mesyanzhinov V.V. Eur. J. Biochem. 2002; 269: 833-841Crossref PubMed Scopus (32) Google Scholar) (GenBank™ accession no. AAD42679) was generated by annealing the oligonucleotide Foldon-5′1 (5′-AGCTTTATATTCCTGAAGCTCCAAGAGATGGGCAAGCTTACGTTCGTAA-3′) with Foldon-5′2(5′-CCATCTTTACGAACGTAAGCTTGCCCATCTCTTGGAGCTTCAGGAATATAA-3′), and Foldon-3′1 (5′-AGATGGCGAATGGGTATTCCTTTCTACCTTTTTATCACCAGCATAAGC-3′) with Foldon-3′2 (5′-GGCCGCTTATGCTGGTGATAAAAAGGTAGAAAGGAATACCCATTCG-3′). The oligonucleotides (Invitrogen) were designed so that HindIII and NotI overhangs (underlined) are created at the 5′ and 3′ ends of the annealed Foldon-5′ and Foldon-3′ fragments, respectively, and cohesive overhangs at the 3′ and 5′ ends. The foldon fragments were co-ligated into HindIII-NotI digested pBluescript (Stratagene) to generate pBSfoldon. To replace the sequence coding for the C-propeptide of the pCα1(I) chain with that coding for foldon, two fragments were generated by PCR, the first extending from an internal BamHI site in the pCα1(I) cDNA to the codon for the last amino acid of the C-telopeptide and the second from the first codon of the foldon cDNA to the NotI site following the stop codon. These fragments were co-ligated into BamHI-NotI-digested pPIC3KpCα1(I) to generate pPIC3Kα1(I)foldon. To delete the C-propeptide, a fragment extending from the internal BamHI site of pCα1(I) cDNA to the codon for the last amino acid of the C-telopeptide followed by a stop codon, and a NotI site was created by PCR and ligated into BamHI-NotI-digested pPIC3KpCα1(I) to generate pPIC3Kα1(I). The pPIC3KpCα1(I), pPIC3Kα1(I)foldon, and pPIC3Kα1(I) constructs were linearized with SalI and transformed into the ArgαAdeβ,his–strain expressing recombinant human prolyl 4-hydroxylase tetramers. Schematic representations of the pCα1(I), α1(I)foldon and α1(I) chains are shown in Fig. 1. PCα2(I), α2(I)foldon, PCα1(I)+PCα2(I), and α1(I)foldon+α2(I)foldon Strains—A P. pastoris expression cassette encoding human pCα2(I) chains was digested from pBLADEIXpCα2(I) (14Nokelainen M. Tu H. Vuorela A. Notbohm H. Kivirikko K.I. Myllyharju J. Yeast. 2001; 18: 797-806Crossref PubMed Scopus (85) Google Scholar) with PmeI-NotI and cloned into pPICZB to generate pPICZBpCα2(I). To replace the C-propeptide of pCα2(I) with foldon, two fragments were generated by PCR, the first extending from an internal AvrII site in the pCα2(I) cDNA to the codon for the last amino acid of the C-telopeptide, and the second encoding foldon as above. These fragments were co-ligated into AvrII-NotI-digested pPICZBpCα2(I) to generate pPICZBα2(I)foldon. The constructs were linearized with PmeI and transformed into the ArgαAdeβ,his–strain to generate the strains PCα2(I) and α2(I)foldon. To study the expression of type I collagen heterotrimers, the linearized pPICZBpCα2(I) and pPICZBα2(I)foldon constructs were transformed into the above strains expressing pCα1(I) and α1(I)foldon chains, respectively. PCα1(III), α1(III)foldon, and α1(III) Strains—To delete the sequence encoding the N-propeptide of proα1(III) chains, a PCR fragment extending from the PmeI site of the alcohol oxidase 5′ sequence of pPICZB to the end of the signal sequence of proα1(III) and followed directly by the sequence coding for the N-telopeptide of proα1(III) until an internal NdeI site was amplified using pPICZBproα1(III) (12Vuorela A. Myllyharju J. Nissi R. Pihlajaniemi T. Kivirikko K.I. EMBO J. 1997; 16: 6702-6712Crossref PubMed Scopus (128) Google Scholar) as a template and a 5′-GCCCATATGAATCATACTG TGCCAAAATAATAGTGGGATGAAGCA-3′ oligonucleotide (with the nucleotides corresponding to the signal sequence and N-telopeptide shown in bold and italics, respectively, and the NdeI site underlined) as the reverse primer. The fragment was cloned into PmeI-NdeI digested pPICZBproα1(III) to generate pPICZBpCα1(III). To change the expression vectors, the proα1(III) and pCα1(III) expression cassettes were digested from the pPICZB vectors with PmeI-NotI and cloned into pPIC3K to generate pPIC3Kproα1(III) and pPIC3KpCα1(III). To replace the C-propeptide of pCα1(III) with foldon, two fragments were generated by PCR, the first extending from an internal AvrII site in the pCα1(III) cDNA to the 3′ end of the C-telopeptide sequence and the second encoding foldon as described above. The fragments were co-ligated into AvrII-NotI-digested pPIC3KpCα1(III) to generate pPIC3Kα1(III)foldon. To delete the C-propeptide, a fragment extending from the internal AvrII site to the 3′ end of the C-telopeptide sequence followed by a stop codon, and a NotI site was created by PCR and ligated into AvrII-NotI-digested pPIC3KpCα1(III) to generate pPIC3Kα1(III). The pPIC3Kproα1(III), pPIC3KpCα1(III), pPIC3Kα1(III)foldon, and pPIC3Kα1(III) constructs were linearized with StuI and transformed into the ArgαAdeβ,his–strain. Culture and Induction of P. pastoris Strains—Cells were cultured in 25-ml shaker flasks in a buffered glycerol complex medium, pH 6.0, with 1 g/liter yeast extract and 2 g/liter peptone. Expression was induced in a buffered minimal methanol medium, pH 6.0, and methanol was added every 12 h to a final concentration of 0.5%. Amino acids were added up to 100 μg/liter as required. Analysis of the Recombinant Collagens—Cells were harvested after a 60-h methanol induction at 30 °C, washed once, and suspended in cold (4 °C) 5% glycerol, 1 mm Pefabloc SC, and 50 mm sodium phosphate buffer, pH 7.4. The cells were broken by vortexing with glass beads, and the lysate was centrifuged at 10,000 × g for 30 min. Aliquots of the soluble fractions were analyzed by SDS-PAGE under reducing conditions followed by Western blotting with polyclonal type I or type III collagen antibodies (Rockland). Further aliquots were digested with pepsin for 2 h at 22 °C or 16 h at 4 °C, the thermal stability of the pepsin-resistant recombinant collagens was studied by digestion with a mixture of trypsin and chymotrypsin for 2 min at various temperatures (28Bruckner P. Prockop D.J. Anal. Biochem. 1981; 110: 360-368Crossref PubMed Scopus (229) Google Scholar), and the samples were analyzed by SDS-PAGE under reducing or nonreducing conditions followed by Coomassie Blue staining or Western blotting as described above. The amounts of the collagen chains were estimated by densitometry of the Coomassie Blue-stained bands using a GS-710 calibrated imaging densitometer (Bio-Rad). Assembly of Stable Type I [α1(I)] 3 and Type III [α1(III)] 3 Homotrimers—It has previously been shown that stable recombinant human type I and type III collagen homotrimers and type I collagen heterotrimers can be produced in P. pastoris by expressing full-length proα1(I) or proα1(III) chains alone or by co-expressing proα1(I) chains with proα2(I) chains in a recombinant strain also expressing human prolyl 4-hydroxylase α2β2 tetramers (12Vuorela A. Myllyharju J. Nissi R. Pihlajaniemi T. Kivirikko K.I. EMBO J. 1997; 16: 6702-6712Crossref PubMed Scopus (128) Google Scholar, 14Nokelainen M. Tu H. Vuorela A. Notbohm H. Kivirikko K.I. Myllyharju J. Yeast. 2001; 18: 797-806Crossref PubMed Scopus (85) Google Scholar). Deletion of the N-propeptides had no effect on the chain assembly, the pCα1(I) and pCα2(I) chains producing heterotrimeric pCcollagen molecules with the correct 2:1 chain ratio (14Nokelainen M. Tu H. Vuorela A. Notbohm H. Kivirikko K.I. Myllyharju J. Yeast. 2001; 18: 797-806Crossref PubMed Scopus (85) Google Scholar). In addition, the expression levels of the type I pCcollagen homotrimers and heterotrimers were 1.5–3-fold relative to those of the corresponding procollagen trimers (14Nokelainen M. Tu H. Vuorela A. Notbohm H. Kivirikko K.I. Myllyharju J. Yeast. 2001; 18: 797-806Crossref PubMed Scopus (85) Google Scholar). To study the effect of replacement of the C-propeptide by the foldon sequence and deletion of the C-propeptide on the assembly of type I and III collagen homotrimers, expression constructs encoding pCα1(I), α1(I)foldon, and α1(I) chains or proα1(III), pCα1(III), α1(III)foldon, and α1(III) chains were transformed by electroporation into a P. pastoris strain expressing active human prolyl 4-hydroxylase. The strains were cultured in buffered glycerol complex medium, and expression was induced in buffered minimal methan