Expression Vector

Abstract

An expression vector including two separately inducible converging promoters P1 and P2, and expression system including such an expression vector and an additional regulator vector, a method of protein expression using such an expression system, and a method of investigating (meta)genome libraries using such an expression system.

1 . An expression vector comprising first and second separately inducible promoters which converge toward each other such that an insertion sequence arranged between said first and second promoters is downstream of each of them so that expression of a DNA sequence cloned into the insertion sequence is placed under the control of said first and second promoters; wherein
 the insertion sequence comprises a polylinker, or a sequence that facilitates integration of DNA sequences by recombination, or both a polylinker and a sequence that facilitates integration of DNA sequences by recombination; and   the expression vector without the insertion sequence comprises at most 3000 base pairs.   
     
     
         2 . An expression vector according to  claim 1 , wherein the expression vector does not code for a regulator of at least one of said first and second promoters. 
     
     
         3 . An expression vector according to  claim 1 , wherein said first promoter is a T7 promoter and said second promoter is an Ara promoter. 
     
     
         4 . An expression vector according to  claim 1 , wherein each expression vector codes for at least one terminator in the reading direction of the corresponding first or second promoter. 
     
     
         5 . An expression vector according to  claim 4 , wherein the at least one terminator comprises a T7 terminator or a terminator for the host RNA polymerase. 
     
     
         6 . An expression vector according to  claim 4 , wherein an additional gene is located between the first promoter and its terminator in the reading direction of the first promoter. 
     
     
         7 . An expression system comprising an expression vector and a regulatory vector, wherein the expression vector comprises separately inducible first and second promoters which converge towards each other such that an insertion sequence arranged between the first and second promoters is downstream of each of them, so that expression of a DNA sequence cloned into the insertion sequence is placed under control of said first and second promoters; wherein
 the insertion sequence comprises a polylinker or a sequence that facilitates integration of DNA sequences by recombination, or both a polylinker and a sequence that facilitates integration of DNA sequences by recombination; and   the regulatory vector codes for a regulator of the first promoter or for a regulator of the second promoter or for both a regulator of the first promoter and a regulator of the second promoter.   
     
     
         8 . An expression system according to  claim 7 , wherein the expression vector without the insertion sequence comprises at most 3000 base pairs. 
     
     
         9 . An expression system according to  claim 8 , wherein the regulatory vector codes for a LacI regulator or for an AraC regulator or for both a LacI regulator and an AraC regulator. 
     
     
         10 . An expression system according to  claim 7 , wherein the regulatory vector additionally contains at least one gene for transfer-RNA of a host organism. 
     
     
         11 . An expression system according to  claim 10 , wherein said gene for transfer-RNA is selected from the group consisting of argU, argW, ileX, gluT, leuW, proL, metT, thrT, tyrU, thrU and argX of  E. coli , which recognize the codons AGG, AGA, AUA, CUA, CCC, GGA or CGG. 
     
     
         12 . An expression system according to  claim 11 , wherein the regulatory vector contains the gene LysS for the T7 lysozyme. 
     
     
         13 . A method of expressing a DNA sequence using an expression system according to  claim 7 , said method comprising:
 (i) optionally transfecting or transforming a suitable host organism with the regulatory vector;   (ii) cloning a DNA sequence or a DNA sequence mixture into the expression vector between the first promoter and the second promoter;   (iii) optionally transfecting or transforming the host organism containing the regulatory vector obtained in (i) with the expression vector containing the DNA sequence or DNA sequence mixture obtained in (ii); and   (iv) inducing expression of the proteins encoded by the DNA sequences by adding the regulator for the first promoter or the regulator for the second promoter or both the regulator for the first promoter and the regulator for the second promoter.   
     
     
         14 . A method according to  claim 13 , wherein the regulator for the first promoter and the regulator for the second promoter are added to spatially separate partial cultures of the obtained host organism which has been transfected or transformed with the regulatory and expression vectors. 
     
     
         15 . A method of screening of a DNA library using an expression system comprising an expression vector and a regulatory vector, wherein the expression vector comprises separately inducible first and second promoters which converge towards each other such that an insertion sequence arranged between the first and second promoters is downstream of each of them, so that expression of a DNA sequence cloned into the insertion sequence is placed under control of said first and second promoters; wherein
 the insertion sequence comprises a polylinker or a sequence that facilitates integration of DNA sequences by recombination, or both a polylinker and a sequence that facilitates integration of DNA sequences by recombination; and   the regulatory vector codes for a regulator of the first promoter or for a regulator of the second promoter or for both a regulator of the first promoter and a regulator of the second promoter; said method comprising expressing the DNA sequence cloned into the insertion sequence using the method of expressing a DNA sequence claimed in  claim 13 .   
     
     
         16 . A method according to  claim 15 , wherein screening is carried out with respect to a catalytic activity of a protein produced by the expression of the DNA sequence cloned into the insertion sequence.