Selective expression of nonsecreted triple-helical and secreted single-chain recombinant collagen fragments in the yeast Pichia pastoris

Pakkanen, Outi; Pirskanen, Asta; Myllyharju, Johanna

doi:10.1016/j.jbiotec.2005.11.012

Cited by 16 publications

(15 citation statements)

References 26 publications

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“…The purified corn-derived rCIa1 was denatured in 6 M guanidine HCl, 50 mM Tris-HCl (pH 8) and 2 mM DTT at 60 C for 45 min followed by six-fold dilution in pH 7.8, 50 mM ammonium bicarbonate buffer for protease (sequencing grade trypsin, Promega, Madison, WI) digestion [3 h at 37 C at an enzyme to substrate ratio of 1:20 (w/w)]. The digest was diluted 2Â with 50% methanol and 0.5% formic acid and the resultant solution was purified and concentrated using a C18 ZipTip (Millipore, Bedford, MA).…”

Section: Characterization Of Rcia1mentioning

confidence: 99%

“…The Pichia-derived non-prolyl hydroxylated rCIa1 and fully prolyl hydroxylated rCIa1 are likewise pepsin-resistant (Figure 3) when expressed as a foldon fusion. 7,37 Without either the foldon or the C-propeptide, the rCIa1 produced from Corn-derived rCIa1(Lanes 1), Pichia-derived non hydroxylated rCIa1(Lanes 2), and Pichia-derived hydroxylated rCIa1chains (Lanes 3) were analyzed without pepsin treatment (A) and after pepsin digestion for 17 h at 4 C (B) by 4-15% SDS-PAGE under reducing conditions followed by silver staining. M stands for a molecular weight marker.…”

Section: Assembly Of Triple-helical Rcia1 Structurementioning

confidence: 99%

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Purification and characterization of a transgenic corn grain‐derived recombinant collagen type I alpha 1

Zhang

Báez

Pappu³

et al. 2009

Biotechnology Progress

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Corn offers advantages as a transgenic host for producing recombinant proteins required at large volumes (1,000's of tons per year) and low cost (less than US$50/kg) by generating them as co-products of biorefining. We describe the purification and characterization of a corn grain-derived mammalian structural protein having such market characteristics: a full length recombinant collagen type I alpha 1 (rCI alpha 1) chain. Material properties of interest are gelation behavior, which would depend on as yet unverified ability of corn to carry out post-translational prolyl hydroxylation and formation of triple helical conformation. The starting material was grain where the expression of rCI alpha 1 had been directed by an embryo-specific promoter. Purification consisted of extraction at low pH followed by membrane and chromatographic steps to isolate rCI alpha 1 for characterization. The amino acid composition and immunoreactivity of CI alpha 1 was similar to that of an analogous native human CI alpha 1 and to rCI alpha 1 produced by the yeast Pichia pastoris. Tandem mass spectrometry confirmed the primary sequence of the corn-derived rCI alpha 1 with 46% coverage. Fragments of the rCI alpha 1 chains were also observed, possibly caused by endogenous plant proteases. The corn-derived rCI alpha 1 had a low level of prolyl hydroxylation (approximately 1% versus 11%) relative to animal-derived CI alpha 1 and folded into its characteristic triple-helical structure as indicated by its resistance to pepsin digestion below its melting temperature of 26(o)C. The 29 amino acid foldon fused to the C-terminus to initiate triple helix formation was not cleaved from the rCI alpha 1 chains, but could be removed by pepsin treatment.

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Section: Characterization Of Rcia1mentioning

confidence: 99%

Section: Assembly Of Triple-helical Rcia1 Structurementioning

confidence: 99%

Purification and characterization of a transgenic corn grain‐derived recombinant collagen type I alpha 1

Zhang

Báez

Pappu³

et al. 2009

Biotechnology Progress

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“…Production of collagen in well-developed prokaryote expression systems has proved challenging because of the lack of prolyl hydroxylase genes [6]. Human collagens have been successfully co-expressed with prolyl hydroxylase genes in yeast [17], and recently these genes were incorporated into E. coli , leading to partial hydroxylation of short collagen-like sequences [18, 19]. In these cases, optimization of expression conditions to balance protein yields and maximum hydroxylation level poses a challenge.…”

Section: Introductionmentioning

confidence: 99%

The influence of specific binding of collagen–silk chimeras to silk biomaterials on hMSC behavior

DesRochers

Qin

et al. 2013

Biomaterials

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Collagen-like proteins in the bacteria Streptococcus pyogenes adopt a triple-helix structure with a thermal stability similar to that of animal collagens, can be expressed in high yield in E. coli and can be easily modified through molecular biology techniques. However, potential applications for such recombinant collagens are limited by their lack of higher order structure to achieve the physical properties needed for most biomaterials. To overcome this problem, the S. pyrogenes collagen domain was fused to a repetitive Bombyx mori silk consensus sequence, as a strategy to direct specific non-covalent binding onto solid silk materials whose superior stability, mechanical and material properties have been previously established. This approach resulted in the successful binding of these new collagen-silk chimeric proteins to silk films and porous scaffolds, and the binding affinity could be controlled by varying the number of repeats in the silk sequence. To explore the potential of collagen-silk chimera for regulating biological activity, integrin (Int) and fibronectin (Fn) binding sequences from mammalian collagens were introduced into the bacterial collagen domain. The attachment of bioactive collagen-silk chimeras to solid silk biomaterials promoted hMSC spreading and proliferation substantially in comparison to the controls. The ability to combine the biomaterial features of silk with the biological activities of collagen allowed more rapid cell interactions with silk-based biomaterials, improved regulation of stem cell growth and differentiation, as well as the formation of artificial extracellular matrices useful for tissue engineering applications.

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“…During the synthesis of recombinant collagens, the majority of procollagens expressed in Pichia pastoris cannot be secreted into the extracellular medium, but are found to accumulate within the ER of the cells even when the authentic signal sequence of the collagen was replaced with the S. cerevisiae α mating factor pre-pro sequence; The latter was reported to be a more efficient signal sequence than the authentic one Pakkanen et al 2006;Vuorela et al 1997). Both the triple-helical conformation and large size of the procollagens ( trimer, ~270 kDa) were found to adversely affect secretion (Pakkanen et al 2006). It is necessary to lyse the cells by external forces to release the procollagens, which are then converted to collagens by the controlled treatment with proteases, commonly human pepsin to remove C-and N-propeptides.…”

Section: Pichia-derived Recombinant Collagens and Gelatinsmentioning

confidence: 99%

“…Since the structure is not a requirement in recombinant gelatins, no co-expression of P4H is needed and the generated gelatins were not hydroxylated . Unlike the collagens, the recombinant gelatins can be secreted into the extracellular medium by introduction of a heterologous signal sequence such as the S. cerevisiae α-mating factor pre-pro sequence Pakkanen et al 2006;Werten et al 1999;. Secretion eliminates the need of cell lysis by physical disruption and reduces the downstream process burden.…”

Section: Pichia-derived Recombinant Collagens and Gelatinsmentioning

confidence: 99%

Purification and characterization of recombinant collagens/gelatins from transgenic corn seeds

Zhang¹

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Selective expression of nonsecreted triple-helical and secreted single-chain recombinant collagen fragments in the yeast Pichia pastoris

Cited by 16 publications

References 26 publications

Purification and characterization of a transgenic corn grain‐derived recombinant collagen type I alpha 1

Purification and characterization of a transgenic corn grain‐derived recombinant collagen type I alpha 1

The influence of specific binding of collagen–silk chimeras to silk biomaterials on hMSC behavior

Purification and characterization of recombinant collagens/gelatins from transgenic corn seeds

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