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Small peptide strengthening mold sequence of strong secretory signal peptide and application thereof

A secretory, signal peptide technology, applied in the field of protein engineering and genetic engineering, to achieve the effect of enhancing the secretion effect

Active Publication Date: 2013-08-21
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although the extracellular secretion expression strategy has significant advantages over other expression methods, there are still many limitations in the widespread use of this strategy in E. coli systems

Method used

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  • Small peptide strengthening mold sequence of strong secretory signal peptide and application thereof
  • Small peptide strengthening mold sequence of strong secretory signal peptide and application thereof
  • Small peptide strengthening mold sequence of strong secretory signal peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1. Application of Small Peptide Enhanced Motif in Secreted Expression of Fructosesidase

[0061] First, the source of the fructosidase Fru6 gene described in this example is Arthrobacter arilaitensis NJEM01, which is the inventor's prior Chinese patent application CN102732456A, and the preservation number of this strain is: CCTCC NO: M2012155.

[0062] All primers involved in this example were synthesized by Yingjun Company, see Table 1. The following primer numbers are uniformly represented by "P" plus a sequence number, for example, primer No. 45 in Table 2, its code is P45, and its number in the sequence table is SEQ ID NO:45.

[0063] The nucleotide sequences of primers and synthetic sequences required for table 1 embodiment 1

[0064]

[0065] Note: The underlined part in the table is the restriction enzyme cutting site.

[0066] (1) Acquisition of the fructosidase FRU6 gene: Using the genome of Arthrobacter arilaitensis NJEM01 as a template, the gene ...

Embodiment 2

[0088] Example 2 Application of Small Peptide Enhancing Motif in Glucanase Secretion and Expression

[0089] (1) Construction of expression vectors with small peptide-enhanced motifs added before different signal peptides at the N-terminal of glucanase BGL

[0090] Same as Example 1, in this example, the fructosidase in Example 1 is replaced by the glucanase gene.

[0091] Among them, the source of the sequence of glucanase BGL: Bacillus subtilis subsp.subtilis6051-HGW, GenBank sequence number: CP003329.1, range: 4011849 to 4012490.

[0092] The gene fragment of glucanase BGL was amplified by PCR reaction with primers P79 and P80, and was prepared into T vector pMD-T-BGL. Overlapping PCR primers P81-P84 or P85-P88 were fused with glucanase BGL and signal peptide ompA or pelB, respectively, and both P84 and P88 had EcoRI restriction sites. P89-P96 upstream primers are used in combination with downstream primers P84 or P88 to design different enhancing motifs to be fused in fr...

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Abstract

The invention belongs to the field of protein engineering and genetic engineering, and relates to a small peptide strengthening mold sequence of a strong secretory signal peptide and an application thereof. The small peptide strengthening mold sequence comprises an amino acid sequence shown in the general formula: M(AlphaXBetaYGamma / AlphaYBetaXGamma)n, wherein the X represents an acidic amino acid; the Y represents a basic amino acid; the Alpha represents 0 to 2 neutral amino acids; the Beta represents 0 to 2 neutral amino acids; the Gamma represents 1 to 10 neutral amino acids; and the n represents 1 to 3. According to the application of the small peptide strengthening mold sequence, the small peptide strengthening mold sequence is use for constructing a carrier for strengthening the secretion capability of a common signal peptide, thereby being a method for improving the common signal peptide for the secretory expression of a foreign protein.

Description

technical field [0001] The invention belongs to the field of protein engineering and genetic engineering, and relates to a class of strongly secreted signal peptide enhancing small peptide modules and applications thereof. Background technique [0002] The protein secretion system is not only the prerequisite for protein sorting, localization and realization of physiological functions, but also an important means for the development of genetic engineering and protein engineering technology. Various prokaryotic and eukaryotic expression systems have been developed for the production of recombinant proteins. Escherichia coli expression system has attracted much attention because of its clear genetic background, simple operation, rapid growth in cheap medium, and easy large-scale fermentation and culture. Remarkable results have been achieved in the field of biotechnology with proteins, such as interferon, insulin, and human serum albumin. [0003] In E. coli, recombinant pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K5/083C07K5/103C07K7/06C07K7/08C07K14/00C12N15/11C12N15/70C12N1/21C12N9/24C12N9/42C12R1/19
CPCC12N15/625C07K7/06C07K7/08C07K14/00C07K2319/02C12N9/2402C12N9/2454C12P21/02C12Y302/01C12Y302/01011
Inventor 何冰芳陈文华米兰吴珊珊朱芸陈珊珊
Owner NANJING TECH UNIV
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