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Process for extracellular secretion of brazzein

A technology of sweet protein and plant, applied in the field of extracellular secretion of plant sweet protein, which can solve the problems of high cost

Pending Publication Date: 2019-02-05
MAGELLAN LIFE SCI PTE LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of these techniques can incur prohibitive costs

Method used

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  • Process for extracellular secretion of brazzein
  • Process for extracellular secretion of brazzein
  • Process for extracellular secretion of brazzein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Codon optimization and gene synthesis:

[0065] The amino acid sequence of the type III form of the plant sweet protein was retrieved from accession P56552 (Ming D et al., FEBS Lett. 355: 106-108 (1994)). The amino acid sequence was reverse-translated into a codon-optimized nucleotide sequence for E. coli. The codon-optimized gene also included an aspartic acid 28 to alanine mutation. This variant was previously shown to exhibit a better sweet taste profile than wild-type type III plant sweetin (Assadi-Porter FM et al., JL.; Arch Biochem Biophys. 2000 Apr 15;376(2):259-65).

[0066] The codon-optimized gene was fused at the N-terminus to a sequence encoding the pelB leader sequence and at the C-terminus to three tandem stop codons.

[0067] The final codon-optimized nucleotide sequence of pelB-phytosweetin is shown in SeqID 1 and the corresponding amino acid translation is shown in SeqID 2.

[0068] The pelB-phytosweetin gene was synthesized by Genscript (...

Embodiment 2

[0069] Embodiment 2: Construction of pET-pelB-plant sweet protein

[0070] The PCR reaction setup is provided in Table 1 below:

[0071] Table 1

[0072]

[0073]

[0074] Table 2

[0075]

[0076] PCR amplification reactions were analyzed on a 1.6% (w / v) agarose gel. Excise about 250bp PCR amplification products corresponding to pelB-plant sweet protein ( figure 2 ), and purified using commercially available kits. The purified product was digested with NdeI and BamHI at 37°C for 4 hours and purified using a PCR spin column kit. It was ligated with pET-28a vector previously digested with NdeI and BamHI and gel purified. The ligation mix was transformed into DH5α competent cells. Transformed cells were plated on LB plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. A single colony was picked from the plate into 5 mL LB broth containing 50 ug / mL kanamycin and grown in an orbital shaker at 37°C and 210 rpm for 16 hours. Plasmid DNA was isolate...

Embodiment 3

[0077] Embodiment 3: Construction of pET-pelB-plant sweet protein (A28D)

[0078] To generate wild type III phytosweetin, amino acid residue 50 of pelB-phytosweetin was mutated from alanine to aspartic acid and named pelB-phytosweetin (A28D).

[0079] The first PCR reaction was set up as follows: final PELBRAZ plasmid, primer PelBLun-FNcoNde (Seq IdNo.3), primer BRAZ-A28DRSOE (Seq Id No.5), Pfu-X reaction buffer, dNTP mix, Pfu-X polymerase And sterile water was used to make the final PCR reaction solution volume to 50 μL. Specific concentrations of the components are provided in Table 3.

[0080] Seq Id No.3:

[0081] 5'-GCGCGCCCATGGCATATGAAATACCTGCTGCCGACCGC-3'

[0082] Seq Id No.5:

[0083] 5'-CACCGCTACGCGCATGTTTTATCCAGTTTACAGTCGTAGTTACATTG GTTCGC-3'

[0084] Table 3: PCR solutions for the first reaction

[0085] components

concentration

Final PELBRAZ plasmid

50ng

PelBLun-FNcoNde (Seq Id No.3)

10 pmol

Braz-A28DRSOE (Seq Id No.5...

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Abstract

The present invention discloses a process for the secretion of brazzein in improved yield.

Description

technical field [0001] The present invention relates to a method for the extracellular secretion of plant sweet proteins from E. coli cells with increased yield and purity. Background technique [0002] Plant sweet protein is a high-potency thermostable sweet protein, which was originally isolated from the fruit of the West African climbing tree Pentadiplandrabrazzeana Baillon (Ming and Hellekant, FEBS Lett. 355: 106-108, 1994). Compared with 2% sucrose aqueous solution, plant sweet protein is 2500 times sweeter than sucrose, and compared with 10% sucrose, plant sweet protein is 500 times sweeter than sucrose. Due to its similar taste profile to sucrose, its water solubility, high temperature and low pH compared to other protein-based natural sweeteners With high stability, plant sweet protein can be conveniently used in baking recipes and by industrial food manufacturers. This stability of plant sweeteners and their sweetness profile make them suitable replacements for cu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A23L2/60C12N15/03C12N1/21
CPCC07K14/43A23L27/31C12N15/70
Inventor A·达基帕提A·V·巴拉甘加哈
Owner MAGELLAN LIFE SCI PTE LTD
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