Production of Bioactive, Post-Translationally Modified, Heterotrimeric, Human Recombinant Type-I Collagen in Transgenic Tobacco

Stein, Hanan; Wilensky, Michal; Tsafrir, Yehuda; Rosenthal, Michal; Amir, Rachel; Avraham, Tal; Ofir, Keren; Dgany, Or; Yayon, Avner; Shoseyov, Oded

doi:10.1021/bm900571b

Cited by 116 publications

(121 citation statements)

References 27 publications

(56 reference statements)

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“…Dual hydroxylation of proline and lysine has not yet been achieved in Pichia pastoris. Coexpression of human prolyl 4-hydroxylase subunits and the lysyl hydroxylase LH3 has been described in tobacco plants, in which recombinant human collagen type I was expressed at up to 200 mg per kg of fresh leaves (Stein et al 2009). …”

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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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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“…Vac-rhCol1 yields were the highest, and molecules were able to form stable triple helical structures that were fully functional in inducing proliferation of human cells. 25 These results highlight that leaf vacuoles are a suitable compartment to store rhCOL1. Another fibrous protein: the spider dragkine silk (DP18) was also fused to a ssVSS: the NPIRL from sporamine.…”

Section: Introductionmentioning

confidence: 78%

“…The genes encoding for rhPCOL1 a 1 and a 2 chains, P4H a, P4H b, and LH3 were expressed in transgenic tobacco plants using different targeting signals to sort to vacuoles (barley aleurain Nt ssVSS MAHARVLLLALAVLATAAVA-VASSSSFADSNPIRPVTDRAASTLA), apoplast or cytosol. 25 Cytoplasm sorted rhCOL1 was not detectable, while apoplast-targeted rhCOL1 yields were very low. Vac-rhCol1 yields were the highest, and molecules were able to form stable triple helical structures that were fully functional in inducing proliferation of human cells.…”

Section: Introductionmentioning

confidence: 95%

Vacuolar deposition of recombinant proteins in plant vegetative organs as a strategy to increase yields

2016

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Delivery of recombinant proteins to vegetative tissue vacuoles was considered inconvenient since this compartment was expected to be hydrolytic; nevertheless there is growing evidence that certain foreign proteins accumulate at high yields in vacuoles. For example avidin, cellulolytic enzymes, endolysin, and transglutaminases were produced at high yields when were sorted to leaf central vacuole avoiding the detrimental effect of these proteins on plant growth. Also, several secretory mammalian proteins such as collagen, a1-proteinase inhibitor, complement-5a, interleukin-6 and immunoglobulins accumulated at higher yields in leaf vacuoles than in the apoplast or cytosol. To reach this final destination, fusions to sequence specific vacuolar sorting signals (ssVSS) typical of proteases or proteinase inhibitors and/or Ct-VSS representative of storage proteins or plant lectins were used and both types of motifs were capable to increase accumulation. Importantly, the type of VSSs or position, either the N or C-terminus, did not alter protein stability, levels or pos-translational modifications. Vacuolar sorted glycoproteins had different type of oligosaccharides indicating that foreign proteins reached the vacuole by 2 different pathways: direct transport from the ER, bypassing the Golgi (high mannose oligosaccharides decorated proteins) or trafficking through the Golgi (Complex oligosaccharide containing proteins). In addition, some glycoproteins lacked of paucimannosidic oligosaccharides suggesting that vacuolar trimming of glycans did not occur. Enhanced accumulation of foreign proteins fused to VSS occurred in several plant species such as tobacco, Nicotiana benthamiana, sugarcane, tomato and in carrot and the obtained results were influenced by plant physiological state. Ten different foreign proteins fused to vacuolar sorting accumulated at higher levels than their apoplastic or cytosolic counterparts. For proteins with cytotoxic effects vacuolar sorted forms yields were superior than ER retained variants, but for other proteins the results were the opposite an there were also examples of similar levels for ER and vacuolar variants. In conclusion vacuolar sorting in vegetative tissues is a satisfactory strategy to enhance protein yields that can be used in several plant species.

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“…This allows them to fold and assemble complex proteins efficiently due to the presence of chaperones and protein disulfide isomerases that catalyze the formation of disulfide bonds, a capability not shared by bacterial production systems. This has proved invaluable for the production of pharmaceuticals, particularly multimeric proteins such as antibodies but also complex technical proteins such as collagen and spider silk (14)(15)(16)(17). The secretory pathway is also where posttranslational modifications (PTMs) are carried out, including glycosylation, γ-carboxylation, β-hydroxylation, amidation, proline hydroxylation, and sulfation (18,19).…”

Section: The Benefits Of Plants As Expression Hostsmentioning

confidence: 99%

Plant Molecular Farming: Much More than Medicines

Tschofen

Knopp

Hood

et al. 2016

Annual Rev. Anal. Chem.

148

103

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Plants have emerged as commercially relevant production systems for pharmaceutical and nonpharmaceutical products. Currently, the commercially available nonpharmaceutical products outnumber the medical products of plant molecular farming, reflecting the shorter development times and lower regulatory burden of the former. Nonpharmaceutical products benefit more from the low costs and greater scalability of plant production systems without incurring the high costs associated with downstream processing and purification of pharmaceuticals. In this review, we explore the areas where plant-based manufacturing can make the greatest impact, focusing on commercialized products such as antibodies, enzymes, and growth factors that are used as research-grade or diagnostic reagents, cosmetic ingredients, and biosensors or biocatalysts. An outlook is provided on high-volume, lowmargin proteins such as industrial enzymes that can be applied as crude extracts or unprocessed plant tissues in the feed, biofuel, and papermaking industries.

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Production of Bioactive, Post-Translationally Modified, Heterotrimeric, Human Recombinant Type-I Collagen in Transgenic Tobacco

Cited by 116 publications

References 27 publications

Recombinant expression of hydroxylated human collagen in Escherichia coli

Recombinant expression of hydroxylated human collagen in Escherichia coli

Vacuolar deposition of recombinant proteins in plant vegetative organs as a strategy to increase yields

Plant Molecular Farming: Much More than Medicines

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