Method of producing a recombinant peptide

A technology for recombinant peptides and peptide cutting, applied in recombinant DNA technology, chemical instruments and methods, peptides, etc., and can solve the problems of expensive synthesis and preparation of peptides

Inactive Publication Date: 2015-02-18
麦德林制药私人有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Synthetically produced VSDLs based on natural VSDL sequences have been used in clinical trials and have proven beneficial for the treatment of disease; however, the synthetic preparation of peptides is an expensive process, especially for peptides that are required on an industrial scale

Method used

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  • Method of producing a recombinant peptide
  • Method of producing a recombinant peptide
  • Method of producing a recombinant peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0178] Embodiment 1 prepares VSDL in escherichia coli

[0179] The amino acid sequence of human VSDL (designated VSDL in the Examples) is:

[0180] Glu-Val-Val-Pro-Pro-Gln-Val-Leu-Ser-Glu-Pro-Asn-Glu-Glu-Ala-Gly-Ala-Ala-Leu-Ser-Pro-Leu-Pro-Glu-Val- Pro-Pro-Trp-Thr-Gly-Glu-Val-Ser-Pro-Ala-Gln-Arg

[0181] (SEQ ID NO:2).

[0182]In this example, the fusion polypeptide approach was utilized in an attempt to attempt the absence of internal arginine (R) and lysine (K) residues in the polypeptide sequence, and the presence of a trypsin cleavage site conveniently provided at the C-terminus Arginine (R) strategy for the study of recombinant expression of VSDL in Escherichia coli ( figure 1 ).

[0183] Preparation of expression constructs

[0184] First, two fusion peptides are designed:

[0185] (i) a single VSDL sequence (designated B72R-VSDL and also referred to herein as "1-mer") fused to a 73 amino acid fusion partner designated B72R, and

[0186] (ii) Three VSDL repeats fu...

Embodiment 2

[0223] Example 2 Recombinant preparation of VSDL 10-mer in Escherichia coli

[0224] Lysates were produced from fermentations of the BR067 E. coli strain described in Example 1 containing the pBRE606 expression construct encoding 10 tandem copies of the VSDL peptide as described in the section "Fed-batch fermentation" of Example 1. B72R-(VSDL) 10 Fusion polypeptides (ie 10-mers).

[0225] Thaw 275 mL of the 10-mer lysate and adjust the pH to pH 8.0 with 1M NaOH. (As described in Example 1) was added to the fusion polypeptide at a mass ratio of 1:120, and digestion was carried out at room temperature for 2 hours. The pH of the digested lysate was adjusted to pH 4.4 with 1M acetic acid to precipitate impurities, and then filtered with a Millistak+Pod Labscale DOHC filter (MD0HC027H2) 0.027m 2 Depth filtering.

[0226] The filtrate was purified by direct loading onto a reverse phase Amberchrom CG300C resin column. The column was flushed with about 15 column volumes of 0.5% ...

Embodiment 3

[0232] Example 3 Recombinant preparation of VSDL 3-link in Escherichia coli

[0233] VSDL lysates were obtained as per Example 2, except that the BR067 E. coli strain described in Example 1 was used containing the pBRE602 expression construct encoding B72R-(VSDL) with 3 tandem copies of the VSDL peptide 3 Fusion polypeptides (ie 3-mers).

[0234] The B72R-(VSDL) 3 The lysate (175 mL) was thawed to a temperature between 15 and 25°C. The pH of the lysate was adjusted to 8.0±0.1 with 1M NaOH. Reconstituted TrypZean was added at 25 μg / mL lysate. Digests were mixed gently for 5 min and then allowed to stand overnight at room temperature. The pH of the digest solution was adjusted to 4.4 ± 0.1 with 1M. The solution was gently stirred using a magnetic stirrer.

[0235] Then pass through Millistak+Pod Labscale D0HC filter (MD0HC027H2) 0.027m 2 The lysate was depth filtered at 4.2 mL / min and then loaded directly onto an Amberchrom CG300C column (16 mm ID column, bed height 7.0 c...

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Abstract

Methods of producing a recombinant peptide, such as vessel dilator peptide (VSDL), are disclosed, including a particular method involving expressing a fusion polypeptide comprising concatemeric repeats of the peptide wherein the peptide is flanked by peptide cleavage sites, and cleaving the fusion polypeptide at the peptide cleavage sites with a cleaving agent(s) so as to release the peptide from the fusion polypeptide. Expression constructs and host cells for producing the fusion polypeptide and purification methods for the recombinant peptide are also disclosed.

Description

technical field [0001] The present invention relates to methods for preparing peptides, especially recombinant peptides. In a particular application, the method involves cleavage of fusion polypeptides comprising tandem copies of the peptides. [0002] quote reference [0003] This patent application claims priority to US Patent Application No. 61 / 612817, filed March 19, 2012. The entire contents of this application are incorporated herein by reference. Background of the invention [0004] Recombinantly produced peptides are of increasing importance both industrially and clinically. Recombinantly produced eukaryotic proteins in various expression systems, such as bacteria, yeast, insect, plant or mammalian cells, provide low-cost alternatives to synthetic peptide preparations. However, recombinant expression is unpredictable and often problematic because many recombinantly expressed peptides are not sufficiently soluble, unstable, misfolded, or inactive, and / or are expre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/62C12N15/70C07K19/00
CPCC07K2319/50C07K2319/00C12N15/62C07K14/575C07K14/58C12P21/06
Inventor 斯坦·巴斯蒂拉斯安吉洛·圭多林本·亨特赛义夫·莱希德雷扎·扎雷尔
Owner 麦德林制药私人有限公司
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