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Recombinant bacteria capable of efficiently expressing GLP-1 analogue and application thereof

A technology of GLP-1 and recombinant bacteria, which is applied in the biological field, can solve the problems of long-term dissolution of inclusion bodies, large renaturation volume, slow growth of protease-deficient strains, and unsuitability for large-scale production, so as to reduce the production of inclusion bodies, Effect of increasing solubility and increasing yield

Active Publication Date: 2021-08-13
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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  • Recombinant bacteria capable of efficiently expressing GLP-1 analogue and application thereof
  • Recombinant bacteria capable of efficiently expressing GLP-1 analogue and application thereof
  • Recombinant bacteria capable of efficiently expressing GLP-1 analogue and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Construction of GLP-1 analog 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-HIS6-...

Embodiment 2

[0053] Example 2 Construction and high expression screening of recombinant engineering bacteria expressing GLP-1 analogs

[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 (7-37...

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 isola...

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Abstract

The invention discloses a recombinant bacteria capable of efficiently expressing glucagon-like peptide 1 (GLP-1) or an analogue thereof. The recombinant bacteria comprise vectors for expressing the GLP-1 or the analogue thereof and vectors for interfering protease expression in the recombinant bacteria. The vectors for interfering protease expression in the recombinant bacteria are expression vectors for interfering protease expression of ClpA, ClpP, ClpQ, ClpX, ClpY, Dcp, PepD and HflB, which are designed by an RNA (Ribonucleic Acid) interference method. Expression of protease in recombinant bacteria is inhibited and interfered, the problem that the GLP-1 analogue is degraded by protease of host bacteria in the fermentation process can be effectively solved, meanwhile, hydrophilic amino acid is added during construction, protein solubility can be remarkably improved, inclusion bodies are reduced, and the proportion of a soluble part is 80%, so that the expression quantity of the GLP-1 analogue is improved to be more than 2g / L. The recombinant bacteria provided by the invention can efficiently express the GLP-1 analogue fusion protein so as to efficiently prepare the GLP-1 analogue.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant bacterium for highly expressing GLP-1 analogues 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(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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