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A kind of recombinant bacteria expressing glp-1 analog and its application

A GLP-1, recombinant bacteria technology, applied in the biological field, can solve the problems of long-term dissolution of inclusion bodies, large volume of renaturation, slow growth of protease-deficient strains, unsuitable for large-scale production, etc. The effect of increasing solubility and increasing yield

Active Publication Date: 2021-11-09
YANGZHOU LIANAO BIOMEDICAL CO LTD +1
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AI Technical Summary

Problems solved by technology

[0006] Some existing studies have developed protease-deficient strains to reduce the degradation of polypeptides or other proteins, but protease-deficient strains often grow slowly and are difficult to quickly carry out high-density fermentation. Some proteases are also required for degrading mutant proteins or providing nutrients, precursors and energy. needs
[0007] CN110128552A, CN110498849A, CN107881187A, CN104592381A, CN108191981A, CN107881187A disclose methods for expressing GLP-1 analogues in Escherichia coli. These methods are all directly introducing recombinant expression vectors into the host without other engineering modifications, and are all in the form of inclusion bodies Express
Inclusion bodies take a long time to dissolve, renature and require a large volume for renaturation, which is not suitable for large-scale production

Method used

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  • A kind of recombinant bacteria expressing glp-1 analog and its application
  • A kind of recombinant bacteria expressing glp-1 analog and its application
  • A kind of recombinant bacteria expressing glp-1 analog and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Construction of GLP-1 analogue expression vector and protease-inhibiting sRNA vector

[0051] Recombinant fusion gene HIS6-Ub-EK-Arg 34 GLP-1 (7-37) 、HIS6-Ub-EK-Arg 34 GLP-1 (9-37) After double digestion with NcoI and EcoRI, they were respectively connected with the vector fragments recovered from PRSFDuet1 after double digestion with NcoI and EcoRI, and the recombinant expression plasmid PRSFDuet1-HIS6-Ub-EK-Arg was obtained by screening 34 GLP-1 (7-37) , PRSFDuet1-HIS6-Ub-EK-Arg 34 GLP-1 (9-37) , and then the recombinant fusion gene HIS6-Ub-EK-Arg 34 GLP-1 (7-37) 、HIS6-Ub-EK-Arg 34 GLP-1 (9-37) After double digestion with NdeI and XhoI, respectively with the plasmid PRSFDuet1-HIS6-Ub-EK-Arg 34 GLP-1 (7-37) , PRSFDuet1-HIS6-Ub-EK-Arg 34 GLP-1 (9-37) The vector fragments recovered after NdeI and XhoI double digestion were ligated, and the double expression plasmid PRSFDuet1-HIS6-Ub-EK-Arg was obtained by screening 34 GLP-1 (7-37) , PRSFDuet1-...

Embodiment 2

[0053] Example 2 Construction of Recombinant Engineering Bacteria Expressing GLP-1 Analogs and High Expression Screening

[0054] Using BL21 (DE3) as the host bacteria, prepare competent cells, and express recombinant plasmid PRSFDuet1-HIS6-Ub-EK-Arg 34 GLP-1 (7-37) , PRSFDuet1-HIS6-Ub-EK-Arg 34 GLP-1 (9-37) and plasmid pBAD-sRNA- clpA , pBAD-sRNA- cLP , pBAD-sRNA- QUR , pBAD-sRNA- wxya , pBAD-sRNA- wxya , pBAD-sRNA- Dcp , pBAD-sRNA- PepD and pBAD-sRNA- wxya Transformed into its competent cells by calcium chloride method to form BL21 (DE3) double plasmid expression strain 1 (PRSFDuet1-HIS6-Ub-EK-Arg 34 GLP-1 (7-37) and pBAD-sRNA- clpA ), 2 (PRSFDuet1-HIS6-Ub-EK-Arg 34 GLP-1 (7-37) and pBAD-sRNA- QUR ), 3 (PRSFDuet1-HIS6-Ub-EK-Arg 34 GLP-1 (7-37) and pBAD-sRNA- cLP ), 4 (PRSFDuet1-HIS6-Ub-EK-Arg 34 GLP-1 (7-37) and pBAD-sRNA- wxya ), 5 (PRSFDuet1-HIS6-Ub-EK-Arg 34 GLP-1 (7-37) and pBAD-sRNA- wxya ), 6 (PRSFDuet1-HIS6-Ub-EK-Arg 34 GLP-1 (7...

Embodiment 3

[0055] Example 3 Expression and Purification of Recombinant GLP-1 Analogs

[0056] Pick single colonies of recombinant dual-plasmid expression strain 3 and dual-plasmid expression strain 11 on the plate, and transfer them to a 500mL Erlenmeyer flask at a ratio of 1:1000 for shaking culture at 37°C after 8 hours of test tube culture. When the OD value reaches 0.6-0.8, use 0.5 mM IPTG was used for induction, and after induction at 25°C for 20 h, the supernatant was centrifuged to obtain bacterial pellets.

[0057] Suspend the cells obtained by centrifugation with 30 mL of protein-purified Buffer A, and use a high-pressure homogenizer to crush the suspended cells to obtain a crude protein sample solution, collect the supernatant by centrifugation, and use Affinity Ni to purify the protein. Different proportions of Buffer B were used for gradient elution to elute the protein containing HIS6, and the eluate was desalted and replaced into the solution Buffer C. Fusion protein is...

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Abstract

The invention discloses a recombinant bacterium for highly expressing glucagon-like peptide 1 (GLP-1) or its analogs. The recombinant bacterium of the present invention includes a carrier for expressing GLP-1 or its analogs and a carrier for interfering with protease expression in the recombinant bacterium. The carrier that interferes with the protease expression in the recombinant bacteria is an expression vector that interferes with the expression of ClpA, ClpP, ClpQ, ClpX, ClpY, Dcp, PepD, and HflB proteases designed by the method of RNA interference. The present invention can effectively solve the problem of GLP-1 analogs being degraded by proteases of host bacteria during the fermentation process by inhibiting and interfering with the expression of proteases in recombinant bacteria. At the same time, adding hydrophilic amino acids during construction can significantly increase protein solubility and reduce inclusion The body is produced, and the soluble part accounts for 80%, thereby increasing the expression level of GLP-1 analogs to more than 2g / L. The recombinant bacterium of the present invention can efficiently express the GLP-1 analog fusion protein, and further efficiently prepare the GLP-1 analog.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant bacterium expressing GLP-1 analogs and its application. Background technique [0002] Human glucagon-like peptide-1 (GLP-1) has medicinal potential in the treatment of type 2 diabetes, and its physiological effects include stimulation of insulin expression, inhibition of glucagon secretion, and reduction of gastric emptying, contributing to glucose levels normalization. With the increasing prevalence of type 2 diabetes worldwide, many GLP-1 derivatives have been developed to enhance the biological activity and drug stability of GLP-1. Its growing demand calls for the development of bioprocesses utilizing recombinant microorganisms for the large-scale production of GLP-1 and its analogues. [0003] Since GLP-1 is a polypeptide hormone whose biological activity does not require eukaryotic post-translational modification, Escherichia coli can be a suitable pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/113C07K14/605C12R1/19
CPCC07K14/605C12N15/1137C12N15/70C12N2310/14
Inventor 彭志恩宋浩柳学伟杨晓瑜巨晓芝郭万成信铭雁
Owner YANGZHOU LIANAO BIOMEDICAL CO LTD
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