High‐level production of human type I collagen in the yeast <i>Pichia pastoris</i>

Nokelainen, Minna; Tu, Hongmin; Vuorela, A.; Notbohm, Holger; Kivirikko, Kari I.; Myllyharju, Johanna

doi:10.1002/yea.730

Cited by 119 publications

(108 citation statements)

References 29 publications

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“…Hydroxylysines are not essential for collagen fibril formation; recombinant type I and III collagens with no hydroxylysine form native-type fibrils in vitro (29,30). Many patients with the kyphoscoliotic type of Ehlers-Danlos syndrome with mutations in the LH1 gene have a virtual absence of hydroxylysine in the collagens of their skin and some other tissues and yet have a relatively mild disease phenotype (7)(8)(9).…”

Section: Discussionmentioning

confidence: 99%

Premature aggregation of type IV collagen and early lethality in lysyl hydroxylase 3 null mice

Rautavuoma

Takaluoma

Sormunen

et al. 2004

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

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102

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Collagens carry hydroxylysine residues that act as attachment sites for carbohydrate units and are important for the stability of crosslinks but have been regarded as nonessential for vertebrate survival. We generated mice with targeted inactivation of the gene for one of the three lysyl hydroxylase isoenzymes, LH3. The null embryos developed seemingly normally until embryonic day 8.5, but development was then retarded, with death around embryonic day 9.5. Electron microscopy (EM) revealed fragmentation of basement membranes (BMs), and immuno-EM detected type IV collagen within the dilated endoplasmic reticulum and in extracellular aggregates, but the typical BM staining was absent. Amorphous intracellular and extracellular particles were also seen by collagen IV immunofluorescence. SDS͞PAGE analysis demonstrated increased mobilities of the type IV collagen chains, consistent with the absence of hydroxylysine residues and carbohydrates linked to them. These results demonstrate that LH3 is indispensable for biosynthesis of type IV collagen and for BM stability during early development and that loss of LH3's functions leads to embryonic lethality. We propose that the premature aggregation of collagen IV is due to the absence of the hydroxylysine-linked carbohydrates, which thus play an essential role in its supramolecular assembly. L ysyl hydroxylase (LH, EC 1.14.11.4, Plod) catalyzes the hydroxylation of lysine residues in -X-Lys-Gly-triplets in collagens and other proteins with collagen-like sequences (1). The hydroxylysine residues formed have two important functions: (i) they serve as attachment sites for carbohydrates, either monosaccharide galactose or disaccharide glucosylgalactose, and (ii) they have an important role in the stabilization of intermolecular collagen crosslinks that provide tensile strength and mechanical stability for the collagen fibrils. The extent of lysine hydroxylation and hydroxylysine glycosylation varies between collagen types. Nearly 90% of all lysines are hydroxylated and hydroxylysines glycosylated in types IV and VI collagen, whereas Ͻ20% of the lysines are hydroxylated in type III collagen (2). The functions of the hydroxylysine-linked carbohydrates are poorly understood, but in the case of fibril-forming collagens, they are thought to regulate the formation and morphology of the fibrils (1, 3).Three human LH isoenzymes have been identified (1, 4-6). Mutations in the LH1 gene lead to the kyphoscoliotic type of Ehlers-Danlos syndrome, which is characterized by scoliosis, joint laxity, skin fragility, ocular manifestations, and severe muscle hypotonia (7-9). Mutations in the LH2 gene have recently been reported in two families with Bruck syndrome, which is characterized by fragile bones, joint contractures, scoliosis, and osteoporosis (10).No mutations have been characterized in the gene for the isoenzyme LH3, and its in vivo functions have not yet been elucidated. This protein differs from the other two lysyl hydroxylase isoenzymes in that it also possesses relatively low levels...

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Section: Discussionmentioning

confidence: 99%

Premature aggregation of type IV collagen and early lethality in lysyl hydroxylase 3 null mice

Rautavuoma

Takaluoma

Sormunen

et al. 2004

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

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102

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“…Human collagen prolyl 4-hydroxylase has been expressed in Escherichia coli (Neubauer et al 2005;Pinkas et al 2011), although with limited activity towards short collagenous substrates. The coexpression of human prolyl 4-hydroxylase subunits and collagen constructs in the yeast Pichia pastoris has 4 enabled the production of prolyl hydroxylated collagen up to 1.5 g per liter of culture (Nokelainen et al 2001). Dual hydroxylation of proline and lysine has not yet been achieved in Pichia pastoris.…”

Section: Introductionmentioning

confidence: 99%

Recombinant expression of hydroxylated human collagen in Escherichia coli

Rutschmann

Baumann

Cabalzar

et al. 2013

Appl Microbiol Biotechnol

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Collagen is the most abundant protein in the human body and thereby a structural protein of considerable biotechnological interest. The complex maturation process of collagen, including essential post-translational modifications such as prolyl and lysyl hydroxylation, has precluded large-scale production of recombinant collagen featuring the biophysical properties of endogenous collagen. The characterization of new prolyl and lysyl hydroxylase genes encoded by the giant virus mimivirus reveals a method for production of hydroxylated collagen. The coexpression of a human collagen type III construct together with mimivirus prolyl and lysyl hydroxylases in Escherichia coli yielded up to 90 mg of hydroxylated collagen per liter culture. The respective levels of prolyl and lysyl hydroxylation reaching 25 % and 26 % were similar to the hydroxylation levels of native human collagen type III. The distribution of hydroxyproline and hydroxylysine along recombinant collagen was also similar to that of native collagen as determined by mass spectrometric analysis of tryptic peptides. The triple helix signature of recombinant hydroxylated collagen was confirmed by circular dichroism, which also showed that hydroxylation increased the thermal stability of the recombinant collagen construct. Recombinant hydroxylated collagen produced in E. coli supported the growth of human umbilical endothelial cells, underlining the biocompatibility of the recombinant protein as extracellular matrix. The high yield of recombinant protein expression and the extensive level of prolyl and lysyl hydroxylation achieved indicate that recombinant hydroxylated collagen can be produced at large scale for biomaterials engineering in the context of biomedical applications.

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“…The range of today's established yeast expression systems includes S. cerevisiae, Kluyveromyces lactis, Pichia pastoris, Yarrowia lipolytica, Arxula adeninivorans, and Hansenula polymorpha (8,17). Polymers such as human collagen and recombinant gelatin have also been produced successfully in several yeast strains (7,12,38,47). The present study describes for the first time the expression of a cyanophycin synthetase gene and the successful production of CGP in yeast.…”

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confidence: 98%

Synthesis and Accumulation of Cyanophycin in Transgenic Strains of Saccharomyces cerevisiae

Steinle

Oppermann‐Sanio

Reichelt

et al. 2008

Appl Environ Microbiol

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Cyanophycin [multi-L-arginyl-poly(L-aspartic acid) (CGP)] was, for the first time, produced in yeast. As yeasts are very important production organisms in biotechnology, it was determined if CGP can be produced in two different strains of Saccharomyces cerevisiae. The episomal vector systems pESC (with the galactoseinducible promoter GAL1) and pYEX-BX (with the copper ion-inducible promoter CUP1) were chosen to express the cyanophycin synthetase gene from the cyanobacterium Synechocystis sp. strain PCC 6308 (cphA 6308 ) in yeast. Expression experiments with transgenic yeasts revealed that the use of the CUP1 promoter is much more efficient for CGP production than the GAL1 promoter. As observed by electrophoresis of isolated CGP in sodium dodecyl sulfate-polyacrylamide gels, the yeast strains produced two different types of polymer: the water-soluble and the water-insoluble CGP were observed as major and minor forms of the polymer, respectively. A maximum CGP content of 6.9% (wt/wt) was detected in the cells. High-performance liquid chromatography analysis showed that the isolated polymers consisted mainly of the two amino acids aspartic acid and arginine and that, in addition, a minor amount (2 mol%) of lysine was present. Growth of transgenic yeasts in the presence of 15 mM lysine resulted in an incorporation of up to 10 mol% of lysine into CGP. Anti-CGP antibodies generated against CGP isolated from Escherichia coli TOP10 harboring cphA 6308 reacted with insoluble CGP but not with soluble CGP, if applied in Western or dot blots.Cyanophycin, also referred to as multi-L-arginyl-poly(L-aspartic acid) or cyanophycin granule polypeptide (CGP), is a nonribosomally synthesized polypeptide consisting of a poly-(aspartic acid) backbone with arginine residues linked to the ␤-carboxyl group of each aspartate by the ␣-amino group (45). CGP is a polydispersed polymer; the molecular size distribution of CGP varies with the producing host strains (1,15,18,20,30,37,52). Due to its branched structure, CGP is not degradable by a wide range of proteinases (45). Biosynthesis of CGP from aspartate and arginine requires only one enzyme, cyanophycin synthetase, which is encoded by cphA (51). CGP is insoluble at neutral pH and under physiological ionic strength, but it is soluble at low (Ͼ3) or high (Ͻ9) pH. Ziegler et al. were the first to observe a water-soluble form of CGP after the heterologous expression of cphA from Desulfitobacterium hafniense strain DSM 10664 in Escherichia coli (52). A detailed study of the solubility behavior of CGP isolated from recombinant E. coli in inorganic salts has been carried out by Füser and Steinbüchel (16). It was shown that the occurrence of the soluble form was not dependent on the origin of cphA or on the host.Recently, several putative applications for CGP and its derivatives have become available, indicating there is a need for its efficient biotechnological production (36,42,43). For the production of CGP at a technical scale, cyanobacteria were shown to be unsuitable due to their low cell...

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High‐level production of human type I collagen in the yeast Pichia pastoris

Cited by 119 publications

References 29 publications

Premature aggregation of type IV collagen and early lethality in lysyl hydroxylase 3 null mice

Premature aggregation of type IV collagen and early lethality in lysyl hydroxylase 3 null mice

Recombinant expression of hydroxylated human collagen in Escherichia coli

Synthesis and Accumulation of Cyanophycin in Transgenic Strains of Saccharomyces cerevisiae

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