Recombined kluyveromyces, construction method and applications thereof

A yeast and recombinant bacteria technology, applied in recombinant DNA technology, chemical instruments and methods, microorganism-based methods, etc., can solve the problems of complex, uneven protein purification, and reduced protein specific activity, and avoid high immunogenicity. , The effect of reducing the number of monosaccharides and not easy to reverse mutation

Active Publication Date: 2011-04-20
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Hyperglycosylation often also leads to inhomogeneity in carbohydrate composition and molecular weight of the recombinant protein product, which complicates protein purification, and the increased carbohydrate moiety also leads to specific activity (units / weight )reduce

Method used

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  • Recombined kluyveromyces, construction method and applications thereof
  • Recombined kluyveromyces, construction method and applications thereof
  • Recombined kluyveromyces, construction method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1, Construction of recombinant Kluyveromyces lactis CGMCC No.2400 inactivated by α-1,6-mannosyltransferase

[0065] 1. Construction of recombinant vector for inactivating α-1,6-mannosyltransferase gene

[0066] Pyrobest enzyme, LA Taq enzyme, dNTPs, restriction endonuclease, T4 ligase, etc. used in the experiment were purchased from Dalian Bao Biological Engineering Co., Ltd., pfu enzyme, kit, DH5α competent cells, primer synthesis, sequencing, etc. were purchased from Provided by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0067] Kluyveromyces lactis ATCC8585 (American Type Culture Collection, American Type Culture Collection, Manassas, Va. 20108 USA), using the genomic DNA as a template to amplify the homology arms on both sides of the α-1,6-mannosyltransferase (OCH1) gene (sequence 1 in the sequence listing), and the homology arms on both sides of OCH1 They are 2kb respectively, and the coding gene of about 1.1kb is missing in the middle. ...

Embodiment 2

[0079] Example 2, Construction of recombinant Kluyveromyces lactis inactivated by α-1,3-mannosyltransferase

[0080] 1. Construction of recombinant vector for inactivating α-1,3-mannosyltransferase gene

[0081] The genomic DNA of Kluyveromyces lactis ATCC8585 was extracted by the glass bead preparation method (A. Adams et al., "Experimental Guidelines for Yeast Genetics Methods", Science Press, 2000), and the genomic DNA was used as a template to amplify α-1,3 -The homology arms on both sides of the mannosyltransferase gene (sequence 2 in the sequence listing), the homology arms on both sides of the α-1,3-mannosyltransferase gene (MNN1) are 1.4kb and 1.5kb respectively, and the middle is deleted The 2.4kb coding gene, the primers used to amplify the homology arms of the upstream flank region of MNN1 are KLMNN01 and KLMNN02, and the primer sequences are: 5′-TAA GCTAGC TGACCAATGACGCATCCGAACT-3' (the underlined part is the NheI recognition site) and 5'-ATT AAGCTT ATGGCACGCTG...

Embodiment 3

[0092] Example 3: Construction of recombinant Kluyveromyces lactis CGMCC No.2401 in which both α-1,6-mannosyltransferase gene and α-1,3-mannosyltransferase gene were inactivated

[0093] Using recombinant Kluyveromyces lactis CGMCC No.2400 as a host, according to the method of Example 2, inactivating the α-1,3-mannosyltransferase gene in the genome of recombinant Kluyveromyces lactis CGMCC No.2400, at 8 Four mutant strains with inactivated α-1,6-mannosyltransferase gene and α-1,3-mannosyltransferase gene were obtained from the clones transfected with pYES2-mnn1 linear fragment. One strain was named KLGE03. The bacterial strain was deposited in the General Microorganism Center of China Committee for Culture Collection of Microorganisms (abbreviated as CGMCC, address: Datun Road, Chaoyang District, Beijing) on ​​March 17, 2008, and the preservation number is CGMCC No.2401.

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Abstract

The invention discloses a recombinant kluyveromyces yeast, a method for constructing the same and an application of the same. The method is to deactivate the alpha-1,6-mannose transferase gene and / or alpha-1,3-mannose transferase gene in a kluyveromyces lactis to get a kluyveromyces lactis strain. The method is to construct a recombinant vector first, then to introduce the recombinant vector intoa kluyveromyces yeast to get a mutant yeast. The recombinant kluyveromyces yeast has two outstanding advantages that: 1), the 80 percent of the coding region of the knockout yeast mannose transferasegene is deleted so reverse mutation is not easy to happen and the stability of a mutant strain is improved obviously; 2) glycoproteins expressed by the recombinant kluyveromyces yeast is free from over glycosylation. The method for constructing the recombinant kluyveromyces yeast of the invention can effectively and successfully recombinant kluyveromyces yeasts, with four positive clones out of eight clones( or a positive rate of 50 percent).

Description

technical field [0001] The invention relates to a recombinant kluyveromyces and its construction method and application. Background technique [0002] Kluyveromyces is a single-celled low-level eukaryotic organism. It has the characteristics of easy cultivation of prokaryotic organisms, low cost of cultivation, fast reproduction, large-scale cultivation and high-density fermentation, and the sugar chain processing system of eukaryotic organisms. , It can also secrete foreign proteins into the culture medium, which is beneficial for purification. Kluyveromyces has been widely used for more than 50 years to produce heterologous proteins of commercial, food or medical importance. So far, the yeast has been used to produce more than 50 proteins from different species. In addition, the medium used by Kluyveromyces to produce heterologous proteins is simple and does not require the addition of methanol like methanol-type yeast (such as: Pichia pastoris), which makes industrial p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C07K14/00C12R1/85C12R1/645
Inventor 刘波吴军马清钧巩新唱韶红
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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