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Method for producing apolipoprotein in plants

A technology of apolipoproteins and plants, applied in the field of producing apolipoproteins, can solve problems such as inapplicable methods and inability to produce apolipoproteins

Inactive Publication Date: 2013-08-28
PHILIP MORRIS PROD SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

All of these problems are particularly acute in the context of the industrial production of recombinant proteins in plants, where multiple or complex steps may render the method unsuitable
[0006] Current apo production systems are not capable of producing apo at levels required for clinical trials or therapeutic applications

Method used

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  • Method for producing apolipoprotein in plants
  • Method for producing apolipoprotein in plants
  • Method for producing apolipoprotein in plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0143] Example 1 - Materials and methods

[0144] Cloning and infiltration

[0145] Nucleic acid constructs comprising nucleotide sequences encoding gamma-zein wild-type gene, fragments and variants thereof were linked to synthetic sequences encoding apolipoprotein A1 Milano, respectively. In the case of using a fragment or variant of γ-zein, the nucleic acid construct further comprises a nucleotide sequence encoding the signal peptide of the native γ-zein 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 apolipoprotein A1 Milano coding sequence was also included in the construct. The coding sequence of apolipoprotein Al Milano 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 ...

Embodiment 2

[0160] Example 2 - Expression Level of Apolipoprotein A1Milano-γ-Zein Fusion Protein

[0161] A gamma-zein-enterokinase-apolipoprotein A1 Milano fusion protein construct (gamma-zein-ApoA1 ) was prepared as described above and transformed into tobacco plants using the Agrobacterium agroinfiltration method. Total protein was extracted and quantified by Western blotting using γ-zein specific antibodies. A control experiment was also carried out using apolipoprotein A1 Milano (ApoA1 ) expressed in the absence of γ-zein under the same conditions. Expression levels from the mean of 3 Agroinfiltration events were as follows:

[0162] For γ-zein-apolipoprotein A1 Milano, the expression level was between about 3 and 6 g γ-zein-apolipoprotein A1 Milano / kg fresh weight.

[0163] For the gamma zein-free apolipoprotein Al Milano, the expression level was between about 2 and 4 g apolipoprotein / kg fresh weight.

[0164] Based on these average results, it was concluded that the expression ...

Embodiment 3

[0165] Example 3 - Analysis of different non-naturally occurring repeat sequence motifs in gamma zein

[0166] A gamma-zein-apolipoprotein A1 Milano fusion construct was prepared using a different non-naturally occurring repeat sequence motif. 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-(PPPEPE)n.

[0167] The constructs were transformed separately into different tobacco plants using the Agrobacterium infiltration method. Total protein was extracted and quantified by Western blotting using γ-zein specific antibodies. Expression levels from the average of 4 Agroinfiltration events were as follows:

[0168] Constructs tested

The expression level

Zein-wild type

1.0

γ-Zein-(PPPVAL)n

1.5

γ-Zein-(PPPVEL)n

0.85

γ-Zein-(PPPAPA)n

1.81

γ-Zein-(PPPEPE)n

1.27

[0169] Results are expressed as relative q...

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Abstract

The present invention provides, in one aspect, a method for producing Apolipoprotein in a plant comprising incubating or growing a plant comprising a nucleic acid construct comprising, consisting or consisting essentially of a nucleic acid sequence encoding an Apolipoprotein fusion protein that comprises a fusion protein partner that induces the formation of a protein body in a plant, preferably, wherein the nucleic acid construct is introduced or infiltrated into the plant prior to the incubating or growing step.

Description

field of invention [0001] The present invention relates to a method for producing apolipoproteins in plants by accumulating apolipoproteins in protein bodies. Nucleic acid sequences, nucleic acid constructs, vectors, expression vectors, etc. for carrying out the methods are also disclosed. Background technique [0002] In healthy people, there is normally a balance between the delivery and removal of cholesterol. When a person has high levels of low-density lipoprotein (LDL) and low levels of high-density lipoprotein (HDL), the imbalance causes more cholesterol to be deposited in the arteries instead of being removed. Atherosclerosis is caused by repeated cholesterol deposits, which lead to narrowing or blockage of arteries. Apolipoprotein A1 (ApoA1) is the major protein component of HDL and plays an important role in cholesterol homeostasis, and studies suggest that ApoA1 and HDL may contribute to a protective role against this disease. [0003] In humans, ApoA1 is produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C07K14/775
CPCC12N15/8257C07K14/775
Inventor K·奥伊什M·派奇
Owner PHILIP MORRIS PROD SA
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