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A cost-effictive method for expression and purification of recombinant proteins in plants

A plant and protein technology, applied in peptide preparation methods, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problem of low expression level, complex expression and purification of ELP-intein fusion, and difficulty in large-scale recovery And other issues

Inactive Publication Date: 2014-04-16
THE CHINESE UNIVERSITY OF HONG KONG
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Problems solved by technology

[0013] Although many previous reports on the expression of ELP fusions of recombinant proteins in plants indicated that ELP fusion proteins could be purified, most of these experiments were performed in tobacco leaves by transient gene expression, which may result in transient hypertrophy of heterologous proteins. accumulation
However, in our initial attempts to express and purify the recombinant protein, human granulocyte colony-stimulating factor (hG-CSF) fused to an ELP-intein tag from a stably transformed tobacco leaf expressed at low levels and was difficult to purify in large quantities. Recovery of target protein (Tian, ​​L. et al., unpublished)
Thus, it appears that ELP fusion expression is quite different from ELP-intein fusion expression, and the expression and purification of ELP-intein fusions in stable plant transformants appears to be more complicated

Method used

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  • A cost-effictive method for expression and purification of recombinant proteins in plants
  • A cost-effictive method for expression and purification of recombinant proteins in plants
  • A cost-effictive method for expression and purification of recombinant proteins in plants

Examples

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Embodiment 1

[0066] Purification of Recombinant Antitumor Proteins from Transgenic Rice Seeds

[0067] In this example, transgenic rice seeds were used as the production system, and the molecular weight (Mw) was about 12.5kD (the protein sequence is shown in figure 1 (c) Pandanus lectin (PAL) as the target protein. PAL shows inhibitory activity against the proliferation of several human cancer cell lines (HepG2, A549, DLD-1, U87 and Capan-2). PAL is expressed as a fusion of an ELP-intein tag, wherein ELP comprises 60 repeats of the "VPGXG" peptide, and the intein protein is cleaved at its C-terminus in response to low pH. The ELP-intein tag is inserted at the N-terminus of PAL, so that after purification of the ELP-intein-PAL fusion protein from rice seeds, intein cleavage can be triggered by lowering the pH of the buffer to isolate the target PAL protein and Ei tag.

[0068] The expression cassette of the ELP-intein-PAL fusion protein is shown in figure 1 (a), the expression casse...

Embodiment 2

[0082] Purification of recombinant human G-CSF from transgenic tobacco

[0083] In this embodiment, transgenic tobacco leaves were used as the production system, while human granulocyte colony-stimulating factor (hG-CSF), an important human cytokine that has been widely used in tumors, was used as the target protein. medicine and infection protection.

[0084] The hG-CSF fusion expression chimera was constructed using the CaMV35S promoter in the binary vector pBI121, and a phaseolin signal peptide was introduced to direct the expressed fusion protein into the secretory pathway of plant cells. The expression cassette is shown in Image 6 . Ubiquitin is introduced to improve the expression of the target protein and will be accurately processed from the fusion protein by endogenous ubiquitin-specific proteases (Ubp) (Tian, ​​L. and Sun, S.S.M., BMC Biotechnol (2011) 11:91). Image 6 The sequence encoding the hG-CSF-intein2-ELP110 fusion protein indicated by brackets is shown...

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Abstract

Downstream purification of recombinant proteins from plant-based samples is performed by elastin-like polypeptides (ELP)-intein fusion expression and purification through inverse phase transition of ELP and cleavage reaction of intein.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application 61 / 425,703, filed December 21, 2010. The content of this document is incorporated herein by reference. technical field [0003] The present invention provides low-cost and efficient methods for expressing and purifying recombinant proteins in transgenic plants. The recombinant protein is expressed as a fusion protein fused with an ELP-intein tag and includes drugs, antibody chains and other useful proteins. It can be produced in different parts of plants such as leaves, seeds, roots, tubers, fruits and cell cultures. Background technique [0004] The use of transgenic plants for large-scale production of pharmaceutical proteins has proven to be an attractive system, which has the advantages of low cost, high yield, easy harvest, and reduced health risks compared with traditional microbial and mammalian bioreactors, and many A valuable recombinant therap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/04C12N5/10C07K1/14
CPCC12N15/62C12N15/8221C12N15/8216C07K14/535C12N15/8257C07K14/42
Inventor 辛世文田莉
Owner THE CHINESE UNIVERSITY OF HONG KONG
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