Use of gelatin-like units

A gelatin-like, purpose-built technology, applied in the protein field, can solve the problems of inability to express the obtained sequence, difficult to predict the spatial structure of the fully artificially designed sequence, and low actual expression.

Inactive Publication Date: 2015-08-05
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the actual effect of these completely redesigned amino acid polymers as fusion carriers is difficult to predict, and there are many problems
For example, 1: Artificially designed sequences, although theoretically contain many hydrophilic amino acids, in view of the complexity of the relationship between protein structure and function, it is difficult to predict the spatial structure of completely artificially designed sequences (such as secondary structure, tertiary structure, etc.), so its potential biological function and immunogenicity are unknown; 2 artificially designed repeat sequences are different from the protein sequences produced by natural evolution, especially those fragments with extremely high repeat sequences, it is difficult For recombinant expression, the actual expression level is often too low to be practically applied
The inventor once used the method provided by David W. Leung et al. (US20080176288) to express polyglutamic acid as a fusion carrier to prolong the protein drug through recombinant expression, but in fact, according to the method provided, it was impossible to express and obtain the claimed sequence

Method used

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Examples

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preparation example Construction

[0112] Preparation of recombinant gelatin-like fusion protein

[0113] The fusion protein of the present invention can be produced by direct synthesis of peptides using solid-phase technology, or the fragments of the protein of the present invention can be chemically synthesized separately and then chemically linked to produce full-length molecules. In a preferred embodiment, the fusion protein of the present invention is produced by recombinant methods.

[0114] The recombinant method to prepare the gelatin fusion protein involves expressing the nucleotides encoding the recombinant target gelatin fusion protein in prokaryotic hosts, eukaryotic hosts, plants or animals, and obtaining the recombinant gelatin-like fusion protein. Any system capable of expressing recombinant proteins, including prokaryotic, eukaryotic, and transgenic animal and plant systems, can be used in the present invention. For example, all the methods for expressing fusion proteins mentioned in US Pat. No...

Embodiment 1

[0159] Example 1: rGLK116 4 protein expression, purification

[0160] 1. GLK116 4 gene cloning

[0161] GLK116 in the present invention 4 The gene is composed of 4 identical monomers (see SEQ ID NO:1 for the sequence) in series, and the monomer is named GLK116 1 , encoding 116 amino acids (see SEQ ID NO: 2 for the sequence), synthesized by Shanghai Yingjun Biotechnology Co., Ltd. (Invitrogen). During synthesis, the α-factor signal peptide sequence of yeast GS115 (position 1-24 in SEQ ID NO:1, with Xho I site) was added to the 5' end, followed by the recognition site of endonuclease DraIII, 3 The 'end has Van91I and EcoRI recognition sites, and is connected to the cloning vector pMD18-T (TaKaRa Company) to construct the plasmid pGLK116 1 -T.

[0162] In order to obtain GLK116 2 Dimer, first the plasmid pGLK116 1 -T was digested with Van91I / Dra III. Electrophoresis was performed on 1% agarose gel, and the target fragment of about 330bp size (ie GLK116 1 ), purified wit...

Embodiment 2

[0178] Example 2: rGLK116 4 Expression, purification and identification of / G-CSF fusion protein

[0179] 1. Synthesis of hG-CSF gene

[0180] The hG-CSF gene (see SEQ ID NO: 4 for the sequence) was synthesized by Shanghai Zeheng Biotechnology Co., Ltd., and cloned into the pMD18-T vector to construct the plasmid pG-CSF-T. The 5' end of G-CSF is the DraIII recognition site, and the 3' end is the EcoRI recognition site.

[0181] 2. Expression plasmid pPIC-GLK116 4 / G-CSF Construction

[0182] Basically the same as Example 1, the construction process of the expression plasmid can be found in image 3 , GLK116 4 / G-CSF DNA coding sequence and mature GLK116 4 See SEQ ID NO:5 and SEQ ID NO:6 for the amino acid sequences of the / G-CSF fusion protein, respectively.

[0183] 3. rGLK116 4 Construction of engineered yeast expressing / G-CSF fusion protein

[0184]pPIC-GLK116 4 / G-CSF transformed methanolic yeast Pichia pastor GS115 (His - ), the plasmid linearization treatment...

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Abstract

The invention relates to a use of a gelatin-like unit and provides a novel recombinant fusion protein. The novel recombinant fusion protein has a basic structure of {gelatin-like protein (GLK)}p-R-{GLK}q, and R represents a protein or a polypeptide having biological functions. GLK is a recombinant gelatin-like protein and has a protein sequence with (Gly-X-Y)n gelatin structural characteristics. Compared with a primitive protein / polypeptide unfused with the GLK fragment, the recombinant gelatin-like fusion protein has higher hydrophility and a longer half life in vivo. The invention also relates to a nucleotide sequence for coding the novel recombinant fusion protein, an expression vector containing the nucleotide sequence, a host cell with the expression vector, and a method for preparing the novel recombinant fusion protein. The invention also relates to a medicinal composition of the novel recombinant fusion protein and a method for treating, preventing or relieving diseases by the medicinal composition.

Description

[0001] The present invention is a divisional application of the invention patent application with application number 200980103870.9. technical field [0002] The present invention relates to the field of protein, more specifically, relates to a new class of recombinant fusion protein with biological activity and longer half-life and its preparation and application. Background technique [0003] Due to the effects of many factors such as kidney and liver and degradation, most clinically used bioactive peptides / proteins are often quickly cleared in the body, and the general half-life is only a few minutes to a few hours. During the course of treatment, a large amount and frequent injections are required to maintain an effective drug concentration, which not only brings pain to the patient, but also reduces the curative effect and increases the toxic and side effects due to the fluctuation of the blood drug concentration. [0004] At present, there are various methods reported ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C07K19/00
CPCC07K14/505C07K14/53C07K14/56C07K14/575C07K2319/31
Inventor 黄岩山周林福陈智
Owner ZHEJIANG UNIV
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