Rhamnose Promoter Expression System

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, and b) 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.

1 . A vector expressible in a host comprising the rhaBAD promoter region of the L-rhamnose operon operably linked to a transcriptional unit comprising:
 a) a nucleic acid sequence which is heterologous to said host, and   b) a prokaryotic signal sequence operably linked to said nucleic acid sequence, whereas said prokaryotic signal sequence is selected from a group consisting of 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.   
     
     
         2 . The vector of  claim 1 , wherein said promoter region is the rhaBAD promoter. 
     
     
         3 . The vector of  claim 2 , wherein said rhaBAD promoter consists of the sequence SEQ ID NO. 1, a sequence complementary thereof and variants thereof. 
     
     
         4 . The vector of  claim 3 , wherein said signal peptides are selected from the group consisting of periplasmatic binding proteins for sugars, amino acids, vitamins and ions, are  E. coli  signal peptides selected from the group consisting of LamB (Maltoporin precursor), MalE (Maltose-binding protein precursor), Bla (Beta-lactamase), OppA (periplasmic oligopeptide-binding protein), TreA (periplasmic trehalase precursor), MppA (periplasmic murein peptide-binding protein precursor), BglX (Periplasmic beta-glucosidase precursor), ArgT (Lysinearginine-ornithine binding periplasmic protein precursor), MalS (Alpha-amylase precursor), HisJ (Histidine-binding periplasmic protein precursor), XylF (D-Xylose-binding periplasmic protein precursor), FecB (dicitrate-binding periplasmic protein precursor), OmpA (outer membrane protein A precursor) and PhoA (Alkaline phosphatase precursor). 
     
     
         5 . The vector of  claim 4 , wherein said transcriptional unit further comprises, a translation initiation region upstream of the initiation point of the translation of said transcriptional unit, said translation initiation region consisting of the sequence AGGAGATATACAT (SEQ ID NO. 2), whereas said translation initiation region is operably linked to said nucleic acid sequence. 
     
     
         6 . The vector of  claim 5 , wherein said transcriptional unit further comprises a transcription termination region which is rrnB transcriptional terminator sequence. 
     
     
         7 . The vector of  claim 6 , wherein said nucleic acid sequence encodes a polypeptide. 
     
     
         8 . The vector of  claim 6 , wherein said nucleic acid sequence encodes an antibody. 
     
     
         9 . The vector of  claim 6 , wherein said nucleic acid sequence encodes a Fab fragment. 
     
     
         10 . The vector of  claim 9 , wherein the heavy and light chain of said Fab fragment are encoded by a dicistronic transcriptional unit, whereas each chain is operably linked to said prokaryotic signal sequence and an identical translation initiation region upstream of the initiation point of the translation of said transcriptional unit. 
     
     
         11 . The vector of  claim 10 , wherein said rhaBAD promoter region and said operably linked transcriptional unit consists of sequence SEQ ID NO. 3, a sequence complementary thereof and variants thereof. 
     
     
         12 . The vector of  claim 10 , wherein said rhaBAD promoter region and said operably linked transcriptional unit consists of sequence SEQ ID NO. 4, a sequence complementary thereof and variants thereof. 
     
     
         13 . The vector of  claim 12 , wherein said vector is an autonomously or self-replicating plasmid, a cosmid, a phage, a virus or a retrovirus. 
     
     
         14 . A process for utilizing the vector of  claim 13 , for regulated heterologous expression of a nucleic acid sequence in a prokaryotic host. 
     
     
         15 . The process of utilizing the vector of  claim 14 , wherein said nucleic acid sequence encodes for a polypeptide. 
     
     
         16 . The process of utilizing the vector of  claim 15 , wherein said polypeptide is a Fab fragment, whereas heavy and light chains of the Fab fragment are expressed in equal amounts. 
     
     
         17 . An isolated and purified nucleic acid sequence expressible in a host comprising rhaBAD promoter region of L-rhamnose operon operably linked to a transcriptional unit comprising:
 a) a nucleic acid sequence which is heterologous to said host, and   b) a prokaryotic signal sequence operably linked to said nucleic acid sequence, whereas said prokaryotic signal sequence is selected from the group consisting of 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.   
     
     
         18 . The isolated and purified nucleic acid sequence of  claim 17 , wherein said promoter region is the rhaBAD promoter. 
     
     
         19 . The isolated and purified nucleic acid sequence of  claim 18 , wherein said rhaBAD promoter consists of sequence SEQ ID NO. 1, a sequence complementary thereof and variants thereof. 
     
     
         20 . The isolated and purified nucleic acid sequence of  claim 19 , wherein said rhaBAD promoter region and said operably linked transcriptional unit consists of sequence SEQ ID NO. 3, a sequence complementary thereof and variants thereof. 
     
     
         21 . The isolated and purified nucleic acid sequence of  claim 19 , wherein said rhaBAD promoter region and said operably linked transcriptional unit consists of sequence SEQ ID NO. 4, a sequence complementary thereof and variants thereof. 
     
     
         22 . Plasmid pBW22-Fab-H. 
     
     
         23 . Plasmid pAKL14. 
     
     
         24 . A prokaryotic host transformed with the vector of  claim 13 . 
     
     
         25 . A prokaryotic host transformed with the isolated and purified nucleic acid sequence of  claim 21 . 
     
     
         26 . A prokaryotic host transformed with the plasmids of  claim 23 . 
     
     
         27 . A method for producing a polypeptide in a host, comprising the steps of:
 a) constructing a vector of  claim 13 ,   b) transforming a prokaryotic host with said vector,   c) allowing expression of said polypeptide in a cell culture system under suitable conditions, and   d) recovering said polypeptide from the cell culture system.   
     
     
         28 . The method of  claim 27 , whereas the polypeptide produced is a Fab fragment, whereas heavy and light chains of the Fab fragment are expressed in said cell culture system in equal amounts. 
     
     
         29 . The method of  claim 28 , whereas expression of said polypeptide is carried out in glycerol containing medium. 
     
     
         30 . A vector expressible in a host comprising a promoter region operably linked to a transcriptional unit comprising:
 a) a nucleic acid sequence which is heterologous to said host, and   b) a translation initiation region upstream of initiation point of the translation of said transcriptional unit, said translation initiation region consisting of sequence AGGAGATATACAT (SEQ ID NO. 2),   
       whereas said translation initiation region is operably linked to said nucleic acid sequence and the expression of said nucleic acid sequence is controlled by said promoter region. 
     
     
         31 . The vector of  claim 30 , wherein said promoter region is rhaBAD promoter region of L-rhamnose operon. 
     
     
         32 . The vector of  claim 31 , wherein said transcriptional unit further comprises a signal sequence operably linked to said nucleic acid sequence. 
     
     
         33 . The vector of  claim 32 , wherein said nucleic acid sequence encodes a polypeptide. 
     
     
         34 . The vector of  claim 32 , wherein said nucleic acid sequence encodes an antibody. 
     
     
         35 . The vector of  claim 32 , wherein said nucleic acid sequence encodes a Fab fragment. 
     
     
         36 . The vector of  claim 35 , wherein heavy and the light chain of said Fab fragment are encoded by a dicistronic transcriptional unit, whereas each chain is operably linked to said signal sequence and said translation initiation region. 
     
     
         37 . A method for producing a polypeptide in a host, comprising the steps of:
 a) constructing a vector of  claim 36 ,   b) transforming a prokaryotic host with said vector,   c) allowing expression of said polypeptide in a cell culture system under suitable conditions, and   d) recovering said polypeptide from the cell culture system.   
     
     
         38 . The method of  claim 37 , whereas the polypeptide produced is a Fab fragment, whereas heavy and light chains of the Fab fragment are expressed in said cell culture system in equal amounts. 
     
     
         39 . The vector of  claim 1 , wherein said signal peptides is selected from the group consisting of periplasmatic binding proteins for sugars, amino acids, vitamins and ions, are  E. coli  signal peptides selected from the group consisting of LamB (Maltoporin precursor), MalE (Maltose-binding protein precursor), Bla (Beta-lactamase), OppA (periplasmic oligopeptide-binding protein), TreA (periplasmic trehalase precursor), MppA (periplasmic murein peptide-binding protein precursor), BglX (Periplasmic beta-glucosidase precursor), ArgT (Lysinearginine-ornithine binding periplasmic protein precursor), MalS (Alpha-amylase precursor), HisJ (Histidine-binding periplasmic protein precursor), XylF (D-Xylose-binding periplasmic protein precursor), FecB (dicitrate-binding periplasmic protein precursor), OmpA (outer membrane protein A precursor) and PhoA (Alkaline phosphatase precursor). 
     
     
         40 . The vector of  claim 1 , wherein said transcriptional unit further comprises, a translation initiation region upstream of initiation point of the translation of said transcriptional unit, said translation initiation region consisting of sequence AGGAGATATACAT (SEQ ID NO. 2), wherein said translation initiation region is operably linked to said nucleic acid sequence. 
     
     
         41 . The vector of  claim 1 , wherein said transcriptional unit further comprises a transcription termination region which is the rrnB transcriptional terminator sequence. 
     
     
         42 . The vector of  claim 1 , wherein said nucleic acid sequence encodes a polypeptide. 
     
     
         43 . The vector of  claim 1 , wherein said nucleic acid sequence encodes an antibody. 
     
     
         44 . The vector of  claim 1 , wherein said nucleic acid sequence encodes a Fab fragment. 
     
     
         45 . The vector of  claim 44 , wherein heavy and light chain of said Fab fragment are encoded by a dicistronic transcriptional unit, whereas each chain is operably linked to said prokaryotic signal sequence and an identical translation initiation region upstream of initiation point of the translation of said transcriptional unit. 
     
     
         46 . The vector of  claim 1 , wherein said rhaBAD promoter region and said operably linked transcriptional unit consists of sequence SEQ ID NO. 3, a sequence complementary thereof and variants thereof. 
     
     
         47 . The vector of  claim 1 , wherein said rhaBAD promoter region and said operably linked transcriptional unit consists of sequence SEQ ID NO. 4, a sequence complementary thereof and variants thereof. 
     
     
         48 . The vector of  claim 1 , wherein said vector is an autonomously or self-replicating plasmid, a cosmid, a phage, a virus or a retrovirus. 
     
     
         49 . A process for utilizing the vector of  claim 1 , for the regulated heterologous expression of a nucleic acid sequence in a prokaryotic host. 
     
     
         50 . The isolated and purified nucleic acid sequence of  claim 17 , wherein said rhaBAD promoter region and said operably linked transcriptional unit consists of sequence SEQ ID NO. 3, a sequence complementary thereof and variants thereof. 
     
     
         51 . The isolated and purified nucleic acid sequence of  claim 17 , wherein said rhaBAD promoter region and said operably linked transcriptional unit consists of sequence SEQ ID NO. 4, a sequence complementary thereof and variants thereof. 
     
     
         52 . A prokaryotic host transformed with the vector of  claim 1 . 
     
     
         53 . A prokaryotic host transformed with the isolated and purified nucleic acid sequence of  claim 17 . 
     
     
         54 . A prokaryotic host transformed with the plasmids of  claim 22 . 
     
     
         55 . A method for producing a polypeptide in a host, comprising the steps of:
 a) constructing a vector of  claim 7 ,   b) transforming a prokaryotic host with said vector,   c) allowing expression of said polypeptide in a cell culture system under suitable conditions, and   d) recovering said polypeptide from the cell culture system.   
     
     
         56 . The method of  claim 55 , wherein expression of said polypeptide is carried out in glycerol containing medium. 
     
     
         57 . The vector of  claim 30 , wherein said transcriptional unit further comprises a signal sequence operably linked to said nucleic acid sequence. 
     
     
         58 . The vector of  claim 30 , wherein said nucleic acid sequence encodes a polypeptide. 
     
     
         59 . The vector of  claim 30 , wherein said nucleic acid sequence encodes an antibody. 
     
     
         60 . The vector of  claim 30 , wherein said nucleic acid sequence encodes a Fab fragment. 
     
     
         61 . A method for producing a polypeptide in a host, comprising the steps of:
 a) constructing a vector of  claim 30 ,   b) transforming a prokaryotic host with said vector,   c) allowing expression of said polypeptide in a cell culture system under suitable conditions, and   d) recovering said polypeptide from the cell culture system