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Corynebacterium glutamicum protein Ncgl0717, and corynebacterium glutamicum cell surface display system and construction method thereof

A Corynebacterium glutamicum, surface display system technology, applied in chemical instruments and methods, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of small types and quantities of anchor proteins, single display system, etc. , to achieve high display efficiency and rich expression types

Active Publication Date: 2021-12-17
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the types and quantities of anchoring proteins of Corynebacterium glutamicum are relatively small at present, and the display system is relatively simple

Method used

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  • Corynebacterium glutamicum protein Ncgl0717, and corynebacterium glutamicum cell surface display system and construction method thereof
  • Corynebacterium glutamicum protein Ncgl0717, and corynebacterium glutamicum cell surface display system and construction method thereof
  • Corynebacterium glutamicum protein Ncgl0717, and corynebacterium glutamicum cell surface display system and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Construction of the Corynebacterium glutamicum wall protein Ncgl0717 surface display vector pEC-EGFP

[0054] (1) Cloning of the wall protein Ncgl0717 gene of Corynebacterium glutamicum

[0055] According to the gene sequence (SEQ ID NO: 2) of Corynebacterium glutamicum wall protein Ncgl0717 (amino acid sequence shown in SEQ ID NO: 1), target protein EGFP gene and Corynebacterium glutamicum plasmid pEC-XK99e (purchased from Novagen ) on the sequence features, design amplification primers:

[0056] P1: 5'-atgAAAGGAGGCCCTTCAGATGAAAACAGAAACTCGACGAGCCCTC-3' (SEQ ID NO: 3);

[0057] P2: 5'-CTTATCGTCATCATCCTTGTAATCGCTCGTGGCAGTTGGTTCCAC-3' (SEQ ID NO: 4).

[0058] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and using P1 and P2 as primers, the sequence of the wall protein Ncgl0717 gene was amplified by PCR. The amplification conditions were: pre-denaturation at 94°C for 5 minutes; followed by 30 more cycles : Denaturation at 94°C fo...

Embodiment 2

[0090] Example 2: Construction of the Corynebacterium glutamicum wall protein Ncgl0717 surface display vector pEC-Ncgl0717-mCherry

[0091] (1) Cloning of the wall protein Ncgl0717 gene of Corynebacterium glutamicum

[0092] According to the gene sequence (SEQ ID NO:2) of Corynebacterium glutamicum wall protein Ncgl0717, target protein mCherry gene and the sequence characteristics on Corynebacterium glutamicum plasmid pEC-XK99e, design amplification primer P1 and P2, by PCR method The gene sequence of the wall protein Ncgl0717 of Corynebacterium glutamicum was amplified. For the specific preparation method, refer to step (1) of Example 1.

[0093] (2) Cloning of target protein mCherry gene

[0094] According to the gene sequence (SEQ ID NO:2) of Corynebacterium glutamicum wall protein Ncgl0717, target protein mCherry gene (SEQ ID NO:17) and the sequence feature on Corynebacterium glutamicum plasmid pEC-XK99e, design amplification primer For P14 and P15, the target protein mC...

Embodiment 3

[0112] Example 3: Construction of the Corynebacterium glutamicum wall protein Ncgl0717 surface display vector pEC-Amy

[0113] (1) Cloning of the wall protein Ncgl0717 gene of Corynebacterium glutamicum

[0114] According to the gene sequence (SEQ ID NO: 2) of Corynebacterium glutamicum wall protein Ncgl0717, design amplification primer, obtain the gene sequence of Corynebacterium glutamicum wall protein Ncgl0717 amplified by PCR method, specific preparation method sees embodiment 1 step (1).

[0115] (2) Cloning of target protein α-amylase gene

[0116] According to the target protein α-amylase (α-amylase EC 3.2.1.1) gene amyE (amyE gene derived from Bacillus subtilis (Bacillus subtilis) 168; sequence shown in SEQ ID NO: 21) design amplification primers P17 and P18 , using the genome of Bacillus subtilis 168 as a template, the gene sequence of the target protein α-amylase was amplified by PCR method, and the preparation method was referred to Example 1:

[0117] P17: 5'-GATT...

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Abstract

The invention discloses a corynebacterium glutamicum protein Ncgl0717, and a corynebacterium glutamicum cell surface display system and a construction method thereof. The corynebacterium glutamicum protein Ncgl0717 has an amino acid sequence as shown in SEQ ID NO: 1. The corynebacterium glutamicum protein Ncgl0717 has a relatively high expression amount and can be used for constructing a corynebacterium glutamicum surface display system with high display efficiency. The corynebacterium glutamicum surface display system is formed by taking the corynebacterium glutamicum wall protein Ncgl0717 as an anchoring protein and fixing a target protein on a surface of a corynebacterium glutamicum cell. An expression variety of an endogenous anchoring protein of the corynebacterium glutamicum surface display system is enriched.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a Corynebacterium glutamicum protein Ncgl0717 and its surface display system and construction method. Background technique [0002] Microbial cell surface display technology is a new type of genetic engineering technology that uses genetic engineering to display target fragments in the form of fusion proteins on the surface of microorganisms through polypeptide fragments (or protein domains). Or polypeptide immobilized on the cell surface, the displayed polypeptide or protein can maintain a relatively independent spatial structure and biological activity. The microbial cell surface display system includes host bacteria, an anchor protein and a target protein, and sometimes a linker sequence is added between the anchor protein and the target protein. Microbial cell surface display has broad application prospects in peptide separation, whole-cell catalysts, whole-cell adsorbents, vacc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/34C12N15/31C12N15/77C12N15/65C12N15/62C12N1/21C12R1/15
CPCC07K14/34C12N15/77C12N15/65C07K14/43595C12N9/2417C12Y302/01001C07K2319/035C07K2319/60Y02E50/10
Inventor 郑穗平林珂瑞韩双艳林影
Owner SOUTH CHINA UNIV OF TECH