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Flavobacterium heparinum expression system

a technology of flavobacterium heparinum and host system, which is applied in the field of new procaryotic expression system of flavobacterium heparinum, can solve the problems of inability to secrete recombinantly produced proteins, inability to glycosylate recombinantly produced proteins, and difficulty in purifying recombinantly produced proteins

Inactive Publication Date: 2005-07-14
BIOMARIN PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text describes a method for using F. heparinum to express recombinant DNA sequences and produce biologically active polypeptides or proteins. The method involves introducing a vector containing the gene sequence of interest into the host cell, which then directs the expression of the protein. The vector can also contain selective markers and a regulated promoter for controlling the expression of the desired gene. The overexpression of genes in the host cell and the suitability of F. heparinum for the production of recombinant proteins are demonstrated by the expression of several enzymes and other proteins.

Problems solved by technology

However, this microorganism has many disadvantages as a host cell including the inability to secrete recombinantly produced proteins, the precipitation of highly expressed recombinantly produced proteins into inclusion bodies within the cell, the inability to glycosylate recombinantly produced proteins and the difficulties faced when purifying a recombinantly produced protein from E. coli.

Method used

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  • Flavobacterium heparinum expression system
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Examples

Experimental program
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Effect test

example 1

F. heparinum Plasmid Systems

[0028] PBBRICm is a derivative of the plasmid pBBR1 with a gene coding for chloramphenicol resistance cloned in. It is a cryptic plasmid isolated from Bordetella bronchiseptica S87. It is further a broad-host-range plasmid known to replicate in E. coli.

[0029] PIBXF2 has the following characteristics: [0030] (1) a bla gene coding for β lactamase (conferring ampicillin resistance)—functional in E. coli; [0031] (2) an ORIT region—allows for the mobilization of the plasmid into a new host (a Sau3 AI 307 bp fragment isolated from plasmid RP4); [0032] (3) a dhfrII gene coding for dihydrofolate reductase type II (conferring trimethoprim resistance)—cloned from plasmid R751 and placed under the control of the hepA promoter—functional in F. heparinum; [0033] (4) a hepA promoter—an 830 bp fragment 5′ of the hepA gene—induced by heparin; [0034] (5) a unique restriction site (for example, a XbaI site)—site for insertion of all homologous and heterologous genes; [00...

example 2

F. heparinum Integration Vector

[0040] The integration vector, pIBXF1, was created by addition of various elements to the parent plasmid, pUC13T.

[0041] The following elements are present on the parent plasmid: [0042] (1) bla gene coding for β lactamase (conferring ampicillin resistance)—functional in E. coli. [0043] (2) ColEI ORI—origin of replication—functional in E. coli. [0044] (3) ORIT region—allows for the mobilization of the plasmid into a new host (a Sau3 AI 307 bp fragment isolated from plasmid RP4).

[0045] The following elements were introduced into the pUC13T plasmid to confer integrative and selective functions in F. heparinum. [0046] (1) 10 kb HindIII fragment—a 10 kb fragment random selected from genomic F. heparinum DNA which was digested with HindIII, and cloned into pUC13T. [0047] (2) dhfrrII gene coding for dihydrofolate reductase type II (conferring trimethoprim resistance)—cloned from plasmid R751 and placed under the control of the hepA promoter—functional only ...

example 3

Introduction of Recombinant DNA into F. heparinum

[0051] Conjugation:

[0052] Plasmids were introduced into the E. coli strain S17-1. S17-1 is a mobilizing strain which provides, in trans, the genes needed for conjugative DNA transfer.

[0053] Overnight cultures of both F. heparinum and E. coli S 17-1 containing the plasmid DNA were subcultured to an OD600 of 0.1 and grown to a mid-log phase of OD600 of 0.5. F. heparinum was grown in MG media supplemented with 1% heparin, 0.02% methionine / histidine, and 2 mM MgSO4 at 23° C. E. coli was grown in Lauria-broth supplemented with 50 μg / mL ampicillin and 10 μg / mL trimethoprim at 37° C.

[0054] 1.75 mL of F. heparinum and 100 μL of E. coli were combined in a 2 mL eppendorf tube and spun at 2500 g for 3 minutes. The cell pellet was resuspended in 100 μL MG and spread over a 0.45 mm membrane (Millipore, catalog number HAWG 025 00) which was placed on an agar petri dish (preheated for 30 minutes at 30° C.) containing LB / MG medium supplemented wi...

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Abstract

This invention is directed to Flavobacterium heparinum for use as a host cell organism for the expression of homologous and heterologous genes.

Description

FIELD OF THE INVENTION [0001] The present invention is in the area of a novel procaryotic expression system and, in particular, a Flavobacterium heparinum host system for the expression of gene products. BACKGROUND OF THE INVENTION [0002] Genetic engineering has made it possible to produce large amounts of heterologous proteins or polypeptides in bacterial cells by means of recombinant expression systems. The expressed heterologous proteins may be mammalian, other eukaryotic, vial, bacterial, cyanobacterial, archeabacterial or of synthetic origin. [0003] The bacterial host cell of choice for expression of recombinant DNA has long been Escherichia coli. However, this microorganism has many disadvantages as a host cell including the inability to secrete recombinantly produced proteins, the precipitation of highly expressed recombinantly produced proteins into inclusion bodies within the cell, the inability to glycosylate recombinantly produced proteins and the difficulties faced when ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/09C12N1/21C12N15/74C12P21/02C12R1/20
CPCC12N15/74
Inventor SU, HONGSHENGSHAO, ZHONGQITKALEC, ANA LYDIABLAIN, FRANCOISEZIMMERMAN, JOSEPH
Owner BIOMARIN PHARMA INC
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