Expression of the sweet protein brazzein in maize for production of a new commercial sweetener

Lamphear, Barry J.; Barker, Donna K.; Brooks, Christopher; Delaney, Donna E.; Lane, Jeffrey R.; Beifuss, Katherine K.; Love, Robert; Thompson, K. K.; Mayor, Jocelyne M.; Clough, Richard W.; Harkey, Robin; Poage, Miranda; Drees, Carol; Horn, Michael E.; Streatfield, Stephen J.; Nikolov, Z̆ivko L.; Woodard, Susan L.; Hood, Elizabeth E.; Jilka, Joseph M.; Howard, John A.

doi:10.1111/j.1467-7652.2004.00105.x

Cited by 75 publications

(42 citation statements)

References 32 publications

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“…The same globulin-1 promoter and barley alpha amylase signal sequence combination had provided higher expression levels of other recombinant proteins in transgenic corn, such as laccase with accumulation level of 51 mg/kg grain 26,29 and brazzein at 400 mg/kg grain. 28 Feasible commercial production would require accumulation above 1 g/kg grain. 30 Backcrossing the transgenic corn with elite germplasm, as has been done before to increase yields, 4 would be a needed future step.…”

Section: Expression Of Cia1 In Cornmentioning

confidence: 99%

“…This signal sequence, combined with the embryo-specific promoter has been shown to achieve high protein expression of other proteins. [26][27][28] The resultant vector was introduced into corn immature embryos using Agrobacterium transformation followed by regeneration to recover transgenic plants, as has been described in the production of a corn grain-derived 44-kDa rCIa1 fragment. 17 …”

Section: Vector Construction and Expression Of Corn Grain-derived Rcia1mentioning

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: Expression Of Cia1 In Cornmentioning

confidence: 99%

Section: Vector Construction and Expression Of Corn Grain-derived Rcia1mentioning

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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“…Advocates of germ targeting have used the embryo-preferred globulin-1 promoter and, as a result, they have reported higher expression levels of recombinant b-glucuronidase (GUS), 6 avidin, 7 fungal laccase, 8 bovine trypsin, 9 and brazzein 10 than when using constitutive or endosperm-preferred promoters. An added advantage for targeting expression to germ is the smaller amount of biomass entering downstream processing 11 because germ is only 11% of the kernel mass.…”

Section: Introductionmentioning

confidence: 99%

Fractionation of transgenic corn seed by dry and wet milling to recover recombinant collagen‐related proteins

Cheng

Glatz

Fox

et al. 2009

Biotechnology Progress

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Corn continues to be considered an attractive transgenic host for producing recombinant therapeutic and industrial proteins because of its potential for producing recombinant proteins at large volume and low cost as coproducts of corn seed-based biorefining. Efforts to reduce production costs have been primarily devoted to increasing accumulation level, optimizing protein extraction conditions, and simplifying the purification. In the present work, we evaluated two grain fractionation methods, dry milling and wet milling, to enrich two recombinant collagen-related proteins; thereby, reducing the amount and type of corn-derived impurities in subsequent protein extraction and purification steps. The two proteins were a full-length human recombinant collagen type I alpha 1(rCIalpha1) chain with telopeptides and peptide foldon to effect triple helix formation and a 44-kDa rCIalpha1 fragment. For each, approximately 60% of the rCIalpha1s in the seed was recovered in the dry-milled germ-rich fractions making up ca. 25% of the total kernel mass. For wet milling, approximately 60% of each was recovered in three fractions accounting for 20-25% of the total kernel mass. The rCIalpha1s in the dry-milled germ-rich fractions were enriched three to six times compared with the whole corn kernel, whereas the rCIalpha1s were enriched 4-10 times in selected wet-milled fractions. The recovered starch from wet milling was almost free of rCIalpha1. Therefore, it was possible to generate rCIalpha1-enriched fractions by both dry and wet milling along with rCIalpha1-free starch using wet milling. Because of its simplicity, the dry milling procedure could be accomplished on-farm thus minimizing the risk of inadvertent release of viable transgenic seeds.

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“…To date, brazzein has been produced in E. coli, 3,4 maize, 5 and Lactococcus lactis. 6 These heterologous productions of brazzein are often complicated by the fact that the protein contains four disulfide bridges and requires a specific N-terminal sequence.…”

mentioning

confidence: 99%

Design and Efficient Soluble Expression of a Sweet Protein, Brazzein and Minor-Form Mutant

Lee¹,

Kong²,

Do³

et al. 2010

Bulletin of the Korean Chemical Society

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Figure 1. The DNA sequence of the synthesized brazzein gene encoding 53 amino acids that combined with the pelB leader sequence and amino acid sequence of the resulted recombinant brazzein. The sequence ends with two TAG and TAA termination codons. Gray box ( ) indicates the sequence of the pelB leader sequence.The demand for non-calorigenic protein-based sweeteners with favorable taste properties is high. Most proteins are tasteless and flavorless, while some proteins elicit a sweet-taste response on the human palate.1 Seven sweet-taste proteins derived from a variety of plants (or rarely from animals) were identified as eliciting a sweet-taste response: thaumatin; monellin; mabinlin; brazzein; neoculin; miraculin; egg white lysozyme. Among them, brazzein possessed better pH and thermal stabilities and a pleasant sweet taste profile. Brazzein was isolated from the fruit of the West African Pentadiplandra brazzeana Baillon plant.2 It is a single-chain polypeptide consisting of 54 amino acid residues, with a corresponding molecular mass of approximately 6.5 kDa. Brazzein is heat-stable, with its sweetness remaining after heating at 98 o C for 2 h and 80 o C for 4.5 h at a pH range of 2.5 -8. This stability may be due to four intramolecular disulfide bonds and the absence of no-free sulfhydryl groups within the molecule. Brazzein water solubility is at least 50 mg/mL. From the point of view of its sweetness, heat stability, high water solubility, and minimum molecular weight, brazzein is currently the superior protein sweetener.Extraction of brazzein from its natural source is expensive and, therefore, not applicable. To date, brazzein has been produced in E. coli, 3,4 maize, 5 and Lactococcus lactis. 6 These heterologous productions of brazzein are often complicated by the fact that the protein contains four disulfide bridges and requires a specific N-terminal sequence. Previous studies for the expression of brazzein from Escherichia coli report that the recombinant brazzein expressed in the cytoplasm of Escherichia coli exists in an insoluble form, involving several steps with a low overall yield: expression as a fusion protein, denaturation and renaturation, oxidation of the cysteines, and cleavage by cyanogen bromide or SUMO protease at an engineered methionine adjacent to the desired N-terminus. To improve production levels of the recombinant soluble brazzein, in the present study, we established a new strategy using the pelB leader sequence of E. coli for brazzein expression. Secretory production of the recombinant proteins by E. coli has several advantages over intracellular production as inclusion bodies. In most cases, targeting protein production to the periplasmic space facilitates downstream processing, folding, and in vivo stability, enabling production of soluble and biologically active proteins at a reduced process cost. When the protein had multiple intramolecular disulfide bonds, it was difficult to achieve proper folding in the cytosol of E. coli since it is highly reducing environment.Neverthel...

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Expression of the sweet protein brazzein in maize for production of a new commercial sweetener

Cited by 75 publications

References 32 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

Fractionation of transgenic corn seed by dry and wet milling to recover recombinant collagen‐related proteins

Design and Efficient Soluble Expression of a Sweet Protein, Brazzein and Minor-Form Mutant

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