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Rhamnose Promoter Expression System

Inactive Publication Date: 2008-08-28
LONZA AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0007]b) a prokaryotic signal sequence operably linked to said nucleic acid sequence, whereas said prokaryotic signal sequence is selected from signal peptides of periplasmatic binding proteins for sugars, amino acids, vitamins and ions and, whereas the expression of said nucleic acid sequence is controlled by said promoter region. Also provided are: the use of said new vector for the regulated heterologous expression of a nucleic acid sequence in a prokaryotic host; an isolated and purified nucleic acid sequence expressible in a host comprising the rhaBAD promoter region of the L-rhamnose operon, a heterologous nucleotide sequence and a prokaryotic signal sequence selected from signal peptides of periplasmatic binding proteins for sugars, amino acids, vitamins and ions; a prokaryotic host transformed with said vector or said isolated and purified nucleic acid sequence; a method for producing a polypeptide in a host using said vector; and a vector comprising a promoter region, a heterologous nucleic acid sequence and a translation initiation region consisting of the sequence AGGAGATATACAT.

Problems solved by technology

However, most heterologous gene expression systems in prokaryotic host systems have relied exclusively on a limited set of bacterial promoters.

Method used

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Examples

Experimental program
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Effect test

example 1

[0112]Construction of Expression Plasmids with Positively Regulated Promoters

[0113]The Escherichia coli W3110 genome was scanned for positively regulated operons. Based on the genomic data which are available on the KEGG database (Kyoto Encyclopedia of Genes and Genomes, http: / / www.genome.ad.jp / kegg / kegg2.html) positively regulated catabolic promoters were identified and analysed for their use in expression plasmids. The promoters should be tightly regulated and induced by a cheap and non-toxic and therefore industrially useful compound. The following promoters of different positively regulated catabolic operons were chosen[0114]prp promoter (propionate inducible)[0115]gutA promoter (glucitol inducible)[0116]melAB2 promoter (melibiose inducible)

[0117]The precise DNA fragments which contain the promoter elements were selected based on the available information on the corresponding regulator binding sites. Chromosomal DNA of Escherichia coli was isolated by the method of Pitcher et al...

example 2

[0120]Construction of Fab Fragment Expression Plasmids

[0121]As an alternative to an IPTG-inducible lac promoter (plasmid pMx9-HuCAL-Fab-H, Knappik et al., 1985, Gene 33, 103-119), different positively regulated expression systems were analysed for their capacity to produce Fab-H antibody fragments. The Fab-H fragment was amplified out of plasmid pMx9-HuCAL-Fab-H by PCR using the primers Fab-5 (5′-aaa cat atg aaa aag aca gct atc-3′) and Fab-3 (5′-aaa aag ctt tta tca gct ttt cgg ttc-3′). The PCR-fragment was cut with NdeI and HindIII and inserted into NdeI / HindIII-cut pBW22 (Volff et al., 1996, Mol. Microbiol. 21, 1037-1047) to create plasmid pBW22-Fab-H (FIG. 1) containing the rhamnose inducible rhaBAD promoter (SEQ ID NO. 1). The same PCR-fragment was inserted into the different expression plasmids with inducible promoters. The resulting Fab-H containing (putative) expression plasmids are pBLL13 containing the prp promoter, pBLL14 containing the gutA promoter and pBLL15 containing t...

example 3

[0122]Expression of Fab Fragment

[0123]Strain W3110 (DSM 5911, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) was transformed with the different expression plasmids. The plasmids were isolated from clones which resulted from the different transformations and checked via restriction analysis. Except plasmid pBLL14 all plasmids had the expected restriction pattern. The re-isolated plasmid pBLL14 showed an altered size and restriction pattern which was suggested to be due to recombination events. Therefore strain W3110 (pBLL14) was not tested in the following assays. The remaining strains were tested for their ability to secrete actively folded Fab-H antibody fragments. This productivity test was performed as described in example 4. The following inducers were added in a concentration of 0.2%

pBW22-Fab-HL(+)-Rhamnose monohydratepBLL13Sodium propionatepBLL15D(+)-Melibiose monohydrateD(+)-Raffinose monohydrateD(+)-Galactose

[0124]The results from the dot...

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Abstract

Vectors expressible in a host that is the rhaBAD promoter region of the L-rhamnose operon operably linked to a transcriptional unit that is:a) a nucleic acid sequence which is heterologouse to the host, andb) a prokaryotic signal sequence operably linked to the nucleic acid sequence.The prokaryotic signal sequence is selected from signal peptides of periplasmatic binding proteins for sugars, amino acids, vitamins and ions. The expression of the nucleic acid sequence is controlled by the promoter region. The vector is used for the regulated heterologous expression of a nucleic acid sequence in a prokaryotic host. This is an isolated and purified nucleic acid sequence expressible in a host is the promoter region of the L-rhamnose operon. There is a method for producing a polypeptide in a host using the vector.

Description

[0001]The present invention concerns vectors for the heterologous expression of nucleic acids encoding e. g. polypeptides such as recombinant proteins in prokaryotic hosts. More specifically, the present invention relates to new vectors expressible in a host comprising the rhaBAD promoter region of the L-rhamnose operon operably linked to a transcriptional unit comprising[0002]a) a nucleic acid sequence which is heterologous to said host[0003]b) a prokaryotic signal sequence operably linked to said nucleic acid sequence, whereas said prokaryotic signal sequence is selected from signal peptides of periplasmatic binding proteins for sugars, amino acids, vitamins and ions and, whereas the expression of said nucleic acid sequence is controlled by said promoter region. Furthermore the invention relates to the use of these vectors for the heterologous expression of nucleic acids encoding e. g. polypeptides.BACKGROUND OF THE INVENTION[0004]Many systems have been described for the heterolog...

Claims

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Application Information

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IPC IPC(8): C12P21/00C12N15/63C12N15/00C07H21/00C12N1/00
CPCC12N15/78C12N15/63C07K16/00C07K2317/14C07K2317/55C07K2317/622C12N15/70
Inventor BRASS, JOHANNKIZIAK, CHRISTOPHKLEIN, JOACHIMOSTENDORP, RALF
Owner LONZA AG
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