Melibiose Operon Expression System

Abstract

New vectors expressible in a host comprising the promoter region of the melibiose operon operably linked to a transcriptional unit that includes nucleic acid sequence which is heterologous to the host. The expression of the nucleic acid sequence is controlled by the promoter region of the melibiose operon. The new vector can be used for the regulated heterologous expression of a nucleic acid sequence in a prokaryotic host. There is an isolated and purified nucleic acid sequence expressible in a host comprising the promoter region of the melibiose operon operably linked to a transcriptional unit that includes a nucleic acid sequence which is heterologous to the host. The expression of the nucleic acid sequence is controlled by the promoter region of the melibiose operon. A prokaryotic host is transformed with the vector or the isolated and purified nucleic acid sequence. There is a method for producing a polypeptide in a host using the vector.

1 . A vector expressible in a host comprising promoter region of the melibiose operon operably linked to a transcriptional unit comprising a nucleic acid sequence which is heterologous to said host, whereas the expression of said nucleic acid sequence is controlled by said promoter region of the melibiose operon. 
     
     
         2 . The vector of  claim 1 , wherein said promoter region is melAB promoter. 
     
     
         3 . The vector of  claim 2 , wherein said melAB promoter is deficient in CRP1 binding site. 
     
     
         4 . The vector of  claim 3 , wherein said melAB promoter deficient in the CRP1 binding site consists of sequence SEQ ID NO. 1, a sequence complementary thereof and variants thereof. 
     
     
         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 signal sequence operably linked to said nucleic acid sequence. 
     
     
         7 . The vector of  claim 6 , wherein said signal sequence is a prokaryotic signal sequence. 
     
     
         8 . The vector of  claim 7 , wherein said prokaryotic signal sequence is selected from signal peptides of periplasmatic binding proteins for sugars, amino acids, vitamins and ions. 
     
     
         9 . The vector of  claim 8 , wherein said transcriptional unit further comprises a transcription termination region which is rrnB transcriptional terminator. 
     
     
         10 . The vector of  claim 9 , wherein said nucleic acid sequence encodes a polypeptide. 
     
     
         11 . The vector of  claim 9 , wherein said nucleic acid sequence encodes an antibody. 
     
     
         12 . The vector of  claim 9 , wherein said nucleic acid sequence encodes a Fab fragment. 
     
     
         13 . The vector of  claim 12 , wherein heavy and light chain of said Fab fragment are encoded by a dicistronic transcriptional unit, whereas each chain is operably linked to a signal sequence and an identical translation initiation region upstream of the initiation point of the translation of said transcriptional unit. 
     
     
         14 . The vector of  claim 13 , wherein said promoter region and said operably linked transcriptional unit consists of the sequence SEQ ID NO. 3, a sequence complementary thereof and variants thereof. 
     
     
         15 . The vector of  claim 13 , wherein said promoter region and said operably linked transcriptional unit consists of the sequence SEQ ID NO. 4, a sequence complementary thereof and variants thereof. 
     
     
         16 . The vector of  claim 15 , wherein said vector is an autonomously or self-replicating plasmid, a cosmid, a phage, a virus or a retrovirus. 
     
     
         17 . A process for utilizing the vector of  claim 16 , for the regulated heterologous expression of a nucleic acid sequence in a prokaryotic host. 
     
     
         18 . The process of utilizing the vector of  claim 17 , wherein said nucleic acid sequence encodes for a polypeptide. 
     
     
         19 . The process of the vector of  claim 18 , wherein said polypeptide is a Fab fragment, whereas heavy and light chains of Fab fragment are expressed in equal amounts. 
     
     
         20 . An isolated and purified nucleic acid sequence expressible in a host comprising promoter region of melibiose operon operably linked to a transcriptional unit comprising a nucleic acid sequence which is heterologous to said host, whereas the expression of said nucleic acid sequence is controlled by said promoter region of the melibiose operon. 
     
     
         21 . The isolated and purified nucleic acid sequence of  claim 20 , wherein said promoter region is melAB promoter. 
     
     
         22 . The isolated and purified nucleic acid sequence of  claim 21 , wherein said melAB promoter is deficient in CRP1 binding site. 
     
     
         23 . The isolated and purified nucleic acid sequence of  claim 22 , wherein said melAB promoter deficient in the CRP1 binding site consists of the sequence SEQ ID NO. 1, a sequence complementary thereof and variants thereof. 
     
     
         24 . The isolated and purified nucleic acid sequence of  claim 23 , wherein said promoter region and said operably linked transcriptional unit consists of sequence SEQ ID NO. 3, a sequence complementary thereof and variants thereof. 
     
     
         25 . The isolated and purified nucleic acid sequence of  claim 23 , wherein said promoter region and said operably linked transcriptional unit consists of sequence SEQ ID NO. 4, a sequence complementary thereof and variants thereof. 
     
     
         26 . Plasmid pBLL15. 
     
     
         27 . Plasmid pAKL15E. 
     
     
         28 . A prokaryotic host transformed with the vector of  claim 16 . 
     
     
         29 . A prokaryotic host transformed with the isolated and purified nucleic acid sequence of  claim 25 . 
     
     
         30 . A prokaryotic host transformed with the plasmids of  claim 27 . 
     
     
         31 . A method for producing a polypeptide in a host, comprising the steps of:
 a) constructing a vector of  claim 16 ,   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.   
     
     
         32 . The method of  claim 31 , 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. 
     
     
         33 . The method of  claim 32 , wherein expression of said polypeptide is carried out in glycerol containing medium. 
     
     
         34 . 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 the sequence AGGAGATATACAT (SEQ ID NO. 2), whereas said translation initiation region is operably linked to said nucleic acid sequence. 
     
     
         35 . The vector of  claim 1 , wherein said transcriptional unit further comprises a signal sequence operably linked to said nucleic acid sequence. 
     
     
         36 . The vector of  claim 1 , wherein said transcriptional unit further comprises a transcription termination region which is the rrnB transcriptional terminator sequence. 
     
     
         37 . The vector of  claim 1 , wherein said nucleic acid sequence encodes a polypeptide. 
     
     
         38 . The vector of  claim 1 , wherein said nucleic acid sequence encodes an antibody. 
     
     
         39 . The vector of  claim 1 , wherein said nucleic acid sequence encodes a Fab fragment. 
     
     
         40 . The vector of  claim 1 , wherein said promoter region and said operably linked transcriptional unit consists of sequence SEQ ID NO. 3, a sequence complementary thereof and variants thereof. 
     
     
         41 . The vector of  claim 1 , wherein said promoter region and said operably linked transcriptional unit consists of the sequence SEQ ID NO.4, a sequence complementary thereof and variants thereof. 
     
     
         42 . The vector of  claim 1 , wherein said vector is an autonomously or self-replicating plasmid, a cosmid, a phage, a virus or a retrovirus. 
     
     
         43 . A process for utilizing the vector of  claim 1 , for the regulated heterologous expression of a nucleic acid sequence in a prokaryotic host. 
     
     
         44 . The isolated and purified nucleic acid sequence of  claim 20 , wherein said promoter region and said operably linked transcriptional unit consists of sequence SEQ ID NO. 3, a sequence complementary thereof and variants thereof. 
     
     
         45 . The isolated and purified nucleic acid sequence of  claim 20 , wherein said promoter region and said operably linked transcriptional unit consists of sequence SEQ ID NO. 4, a sequence complementary thereof and variants thereof. 
     
     
         46 . A prokaryotic host transformed with the vector of  claim 1 . 
     
     
         47 . A prokaryotic host transformed with the isolated and purified nucleic acid sequence of  claim 20 . 
     
     
         48 . A prokaryotic host transformed with the plasmids of  claim 26 . 
     
     
         49 . A method for producing a polypeptide in a host, comprising the steps of:
 a) constructing a vector of  claim 10 ,   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   
     
     
         50 . The process of the vector of  claim 49 , wherein said polypeptide is a Fab fragment, whereas heavy and light chains of the Fab fragment are expressed in equal amounts. 
     
     
         51 . The method of  claim 49 , whereas expression of said polypeptide is carried out in glycerol containing medium.