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Stably Maintained Multiple Copies of at Least Two Orf in the Same Orientation

a technology of multiple copies and orientation, applied in the field of building stable cells, can solve the problems of unstable plasmids, often lost from the host cell, and instability of multiple copies of the gene, and achieve the effect of stable integration

Inactive Publication Date: 2008-02-21
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006] It has now been found that at least two copies of the “same” gene can be stably integrated on the host cell chromosome in parallel tandem by introducing changes into the DNA sequence of at least one of the gene copies which does not change the amino acid sequence of the resulting protein encoded by the changed DNA sequence. In this way it is possible to have multiple copies of a

Problems solved by technology

However, plasmids are unstable and are often lost from the host cells if there is no selective pressure during the cultivation of the host cells.
A problem associated with integrating several copies of a gene into the chromosome of a host cell is instability of the multiple copies of the gene.
This, however, would lead to the described instability of the construct.
Such a construct will normally be unstable when integrated on the chromosome due to the above described recombination taking place.

Method used

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  • Stably Maintained Multiple Copies of at Least Two Orf in the Same Orientation
  • Stably Maintained Multiple Copies of at Least Two Orf in the Same Orientation

Examples

Experimental program
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Effect test

example 1

Synthetic DNA sequences encoding protease 10R

[0142] Protease 10R is a proteolytic enzyme produced by Nocardiopsis prasina NRLL 18262.

[0143] The DNA sequence given as SEQ ID NO: 1 is a synthetic DNA sequence encoding the signal peptide coding region from the Bacillus clausii alkaline protease gene, Savinase®, followed in frame by a synthetic DNA sequence encoding the pro+mature parts of the 10R protease. The synthetic DNA sequence is changed compared to the Nocardiopsis wildtype sequence, but it still encodes the wildtype 10R enzyme. A synthetic gene can be constructed by PCR assembly of overlapping oligonucleotides in various methods described eg. by Stemmer et al, Gene 164, pp-49-53, 1995; Dillon and Rossen, BioTechniques 9, 298-300, 1990; Prodromou and Pearl, Protein Engineering 5, 827-829, 1992; Chn et al., Journal of Amarical Chemical Sociaty 11, 8799-8800, 1994 and others.

[0144] Genes of the desired sequence can also be purchased from commercial companies like DNA2.0 inc., 1...

example 2

Construction of strain Bacillus subtilis ppP715-4, containing a chromosomally integrated copy of a synthetic gene encoding the protease 10R enzyme

[0147] The use of pDG268-derived plasmids for introduction of aprL expression cassettes into the amyE locus of B. subtilis in single copy has been described in details (Widner, B., Thomas, M., Sternberg, D., Lammon, D., Behr, R., and Sloma, A. (2000) Development of marker-free strains of Bacillus subtilis capable of secreting high levels of industrial enzymes. Journal of Industrial Microbiology & Biotechnology, 25, 204-212.; Widner, W., Sloma, A., Thomas, M. D. (2003) Methods for producing a polypeptide in a Bacillus cell. United States Patent Application Publication US 2003 / 0170876 A1). FIG. 25 in patent application US 2003 / 0170876 specifically illustrates a plasmid containing a particular composite promoter, including the cryIIIA promoter and mRNA stabilizing region. This plasmid was used as a vector plasmid to make a construct, in whic...

example 3

Construction of a plasmid, pSJ6869, carrying SEQUENCE ID NO: 1 encoding protease 10R

[0148] pSJ5801

[0149] This is a pUC19 (Yanisch-Perron et al., 1985, Gene 33(1): 103-119) based plasmid carrying the transcriptional terminator from the B. licheniformis alpha-amylase gene, amyL. It was made by PCR amplification with:

Primer 1:(SEQ ID NO: 9)5′-GACTCTGCAGCCGCGGACGCGTGCTAGCGGCCGCGTCGACTAGAAGAGCAGA-GAGGACGG-3′andPrimer 2:(SEQ ID NO: 10)5′-GACTAAGCTTATCGATGATCAAGATCTCAACGAAATTTATAAGACGGGC-3′

using chromosomal DNA of B. licheniformis as template, digestion of the PCR fragment with HindIII and PstI, and ligation into HindIII+PstI digested pUC19. The ligation mixture was transformed into E. coli SJ2 (Diderichsen et al., 1990, J. Bacteriol. 172(8): 4315-4321) by electroporation, selecting ampicillin resistance. 2 transformants, harbouring plasmids with the correct sequence of the PCR amplified terminator segment, were kept as SJ5801 (SJ2 / pSJ5801) and SJ5802 (SJ2 / pSJ5802).

[0150] pSJ6074

[01...

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Abstract

The present invention relates to a method for constructing a host cell expressing a polypeptide of interest from at least two ORF's stably integrated onto the chromosome of the host cell comprising the steps of: a) providing the at least two ORF's encoding the same polypeptide, wherein the at least two DNA sequences of the ORF's differ in at least one position; b) integrating the at least two ORF's in the same orientation on the host cell chromosome.

Description

TECHNICAL FIELD [0001] A method for constructing a stable cell expressing a gene of interest and comprising at least two copies of said gene on the chromosome. BACKGROUND ART [0002] In the industrial production of polypeptides it is of interest to achieve a product yield as high as possible and to be able to control the said expression. One way to increase the yield is to increase the copy number of a gene encoding a polypeptide of interest. This can be done by placing the gene on a high copy number plasmid. However, plasmids are unstable and are often lost from the host cells if there is no selective pressure during the cultivation of the host cells. Another way to increase the copy number of the gene of interest is to integrate it into the host cell chromosome in multiple copies. It has previously been described how to integrate a gene into the chromosome by double homologous recombination without using antibiotic markers (Hone et al., Microbial Pathogenesis, 1988, 5: 407-418); in...

Claims

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Application Information

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IPC IPC(8): C12P21/00C07H21/00C12N1/00C12N9/52C12N15/67C12N15/90
CPCC12N15/67C12N15/902C12N9/52C12N2830/20C12N2800/40
Inventor JORGENSEN, STEEN TROELSPEDERSEN, POUL ERIK
Owner NOVOZYMES AS