Method for expressing deoxyribonuclease in plants

A plant, nucleic acid sequence technology, applied in the fields of biochemical equipment and methods, plant peptides, chemical instruments and methods, etc., can solve problems that cannot be easily scaled to commercial levels

Inactive Publication Date: 2013-08-21
PHILIP MORRIS PROD SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Additionally, existing DNase expression systems utilize mammalian cells as expression hosts, which means that downstream processing (e.g., purification and amplification) of the expressed protein cannot be easily scaled up to commercial levels

Method used

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  • Method for expressing deoxyribonuclease in plants
  • Method for expressing deoxyribonuclease in plants
  • Method for expressing deoxyribonuclease in plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0138] Example 1 - Materials and methods

[0139] Cloning and infiltration

[0140] The nucleic acid constructs comprising the nucleotide sequences encoding the gamma-zein wild-type gene, its fragments and variants are respectively linked to the synthetic sequences encoding the DNase. In the case of using a fragment or variant of γ-zein, the nucleic acid construct also comprises a nucleotide sequence encoding a natural γ-zein signal peptide at the 5′ end, if it is present in the fragment or variant words that exist in the body. For some experiments, a synthetic nucleic acid sequence encoding a linker comprising a protease cleavage site between the gamma-zein coding sequence and the DNase coding sequence was also included in the construct. The coding sequence of the DNase has been optimized for expression in plants. The nucleic acid construct is cloned into a vector at a site where expression of the nucleic acid construct in tobacco plant cells is driven by the minimal 35S p...

Embodiment 2

[0149] Example 2 - Expression Levels of γ-Zein-DNase I Fusion Protein

[0150] A gamma-zein-DNase I fusion protein construct (gamma-zein-DNase I) was prepared as described above and transformed into tobacco plants using the Agroinfiltration method. Total protein was extracted and quantified by Western blotting using γ-zein specific antibodies. A control experiment (hDNase I) was also performed using DNase I expressed under the same conditions without the gamma-zein tag. The expression levels from the mean values ​​of Agroinfiltration events were as follows:

[0151]

[0152] FW = net weight (free weight). Based on these results, it was concluded that the expression of γ-zein-hDNase I was higher than that of human DNase I in the absence of γ-zein.

Embodiment 3

[0153] Example 3 - Analysis of different non-naturally occurring repeat sequence motifs in gamma zein

[0154] γ-Zein-DNase I fusion constructs were made using different non-naturally occurring repeat sequence motifs in γ-zein. The following constructs were used: γ-zein peptide only (zein-wild type); γ-zein-(PPPVAL)n; γ-zein-(PPPVEL)n; γ-maize prolamin-(PPPAPA)n; and gamma-zein-free.

[0155] The constructs were transformed separately into different tobacco plants using the Agrobacterium infiltration method. Three independent transformations were performed. Total protein was extracted and quantified by Western blotting using γ-zein specific antibodies. Expression levels from the mean of 3 Agroinfiltration events were as follows:

[0156] construct

[0157] Results are expressed as relative quantification compared to γ-zein wild type. Unexpectedly, human DNase I was expressed at the highest level in the absence of γ-zein. Other non-naturally occurring repeat mot...

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Abstract

The present invention provides, in one aspect, a method for expressing DNase in a plant. The method comprises growing a plant that has been transformed with a nucleic acid construct comprising a nucleic acid sequence encoding DNase under the control of a regulatory nucleotide sequence that regulates the transcription of said nucleic acid sequence in said plant.

Description

field of invention [0001] The present invention relates to a method for expressing recombinant deoxyribonucleases, suitably human DNase I, in plants. Nucleic acid sequences, nucleic acid constructs, vectors, expression vectors, etc. for carrying out the methods are also disclosed. Background technique [0002] Deoxyribonucleases (DNases) are phosphodiesterases that hydrolyze polydeoxyribose nucleic acids and are known to occur in several molecular forms. Based on their biochemical properties and enzymatic activity, DNase proteins have been divided into two types: DNase I and DNase II. The DNase I protein has a near-neutral pH optimum (a necessary requirement for divalent cations) and generates 5'-phosphate nucleotides when hydrolyzing DNA. DNase II proteins exhibit an acidic pH optimum, can be activated by divalent cations, and generate 3'-phosphate nucleotides upon hydrolysis of DNA. [0003] DNase proteins from various species have been described, including bovine DNase...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
CPCC12N15/8243C12N15/8257C07K14/415C12N9/22C12N15/8251
Inventor K·奥伊什M·派奇
Owner PHILIP MORRIS PROD SA
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