Nucleic acids for cloning and expressing multiprotein complexes

Abstract

The present invention relates to a nucleic acid containing at least one homing endonuclease site (HE) and at least one restriction enzyme site (X) wherein the HE and X sites are selected such that HE and X result in compatible cohesive ends when cut by the homing endonuclease and restriction enzyme, respectively, and the ligation product of HE and X cohesive ends can neither be cleaved by the homing endonuclease nor by the restriction enzyme. Further subject-matter of the present invention relates to a vector comprising the nucleic acid of the present invention, host cells containing the nucleic acid and/or the vector; a kit for cloning and/or expression of multiprotein complexes making use of the vector and the host cells, a method for producing a vector containing multiple expression cassettes, and a method for producing multiprotein complexes. The invention also relates to a methods of assembling multiple single vectors (“vector entities”) into fusion vectors and to method of disassembling a fusion vector containing multiple of such vector entities into single vectors. The invention is also directed to fusion vectors containing multiple vector entities.

1 . A nucleic acid comprising a multiple integration element (MIE) having the following sequence elements: 
       
         
           
           
               
               
           
         
         wherein 
         HE is a homing endonudease site selected from the group consisting of a I-CeuI site and a PI-SceI site; 
         Prom represents a promoter; 
         rbs represents a ribosome binding site; 
         term represents a terminator; 
         and wherein the HE and BstX sites are selected such that HE and BstXI result in compatible cohesive ends when cut by the homing endonuclease and the BstXI restriction enzyme, respectively, and the ligation product of HE and BstXI cohesive ends can neither be cleaved by the homing endonoclease nor the restriction enzyme. 
       
     
     
         2 . The nucleic acid of  claim 1  further comprising the nucleotide sequence of SEQ ID NO: 1. 
     
     
         3 . The nucleic add of  claim 1  claims further comprising at least one site for integration of the nucleic acid of  claim 1  into a vector or host cell. 
     
     
         4 . A vector comprising the nucleic acid of  claim 1 . 
     
     
         5 . The vector of  claim 4  further comprising at least one recognition sequence for a site-specific recombinase, preferably a LoxP imperfect inverted repeat or a Tn7 attachment site. 
     
     
         6 . The vector of  claim 4  comprising a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17. 
     
     
         7 . The vector of  claim 6  comprising more than one of the sequence elements of the nucleic acid as defined in  claim 1  and containing more than one recognition sequence for a site-specific recombinase. 
     
     
         8 . The vector of  claim 7  comprising the sequence of SEQ ID NO: 18. 
     
     
         9 . The vector of  claim 4  wherein the vector is a virus. 
     
     
         10 . The vector of  claim 9  wherein the virus is a baculovirus. 
     
     
         11 . A host cell comprising the nucleic acid of  claim 1 . 
     
     
         12 . A host cell comprising the vector of  claim 4 . 
     
     
         13 . A kit for cloning and/or expression of multiprotein complexes containing at least one vector of  claim 4  together with at least one host cell suitable for the propagation of said vector(s). 
     
     
         14 . A method for assembling n vector entities each containing a multiple integration element as defined in  claim 1  into 1 to (n−1) fusion vectors wherein said fusion vector(s) contain(s) 2 to n of said vector entities comprising the steps of:
 (1) contacting said n vector entities each containing a site-specific recombination site and an individual resistance marker different from the resistance markers of the other vector entities with a recombinase specific for said site-specific recombination site so as to generate a mixture of fusions of the vector entities comprising 2 to n of said vector entities, 
 (2) transforming said mixture into host cells; 
 (3) culturing one or more sample(s) of the transformed cells in the presence of an appropriate combination of antibiotics for selecting one or more desired fusion vector(s) containing 2 to n vector entities; 
 (4) obtaining n single clones of transformed cells from the culture obtained in step (3) in which these were viable in the presence of the respective combination of antibiotics; and 
 (5) culturing n samples of each of said n single clones in the presence of each of n antibiotics specific for the n individual resistance markers present in said n vector entities; 
 wherein n is an integer of at least 3. 
 
     
     
         15 . The method of  claim 14  wherein (n−1) of the vector entities to be fused each contains a further selectable marker different from the resistance markers such that only host cells transformed with fusions between the vector entity not containing the further selectable marker and one or more of the vector entities containing the selectable marker are viable in step (3). 
     
     
         16 . The method of  claim 15  wherein (n−1) of the vector entities contain a conditional origin of replication making the propagation of said vector entities dependent on the presence or absence of a specific gene in the host cells. 
     
     
         17 . The method of  claim 16  wherein the host cells are bacteria, preferably  E. coli , the origin of replication is R6Kγ or a derivative thereof and the bacteria are pir − . 
     
     
         18 . The method of  claim 14  wherein each of the n vector entities contains one or more genes of interest, preferably within an expression cassette. 
     
     
         19 . A method of disassembling a fusion vector containing n vector entities each containing a multiple integration element (MIE) as defined in  claim 1  into one or more desired fusion vectors selected from the group consisting of fusion vectors containing 2 to (n−1) vector entities or into one or more of said single vector entities each containing a multiple integration element (MIE) as defined in  claim 1 , wherein in said fusion vector containing n vector entities said n vector entities are separated from each other by n site-specific recombination sites, and each vector entity contains an individual resistance marker different from the resistance markers of the other vector entities, comprising the steps of:
 (A) contacting the fusion vector containing n vector entities each containing a multiple integration element (MIE) as defined in  claim 1  with a recombinase specific for said site-specific recombination sites in order to generate a mixture of fusions of the vector entities comprising 2 to (n−1) of said vector entities and single vector entities; 
 (B) transforming said mixture into host cells; and 
 (C) culturing one or more sample(s) of the transformed cells in the presence of:
 (C1) an appropriate combination of antibiotics for selecting one or more desired fusion vecter(s) containing 2 to (n−1) vector entities; and/or 
 (C2) a single appropriate antibiotic for selecting a desired single vector entity; 
 
 (D) obtaining n single clones of transformed cells from the sample of the transformed cells in which the single clones of transformed cells were viable in the presence of the respective antibiotic or combination of antibiotics, respectively, and 
 (E) culturing n samples of each of said n single clones of transformed cells in the presence of each of n antibiotics specific for the n individual resistance markers present in said n vector entities; 
 wherein n is an integer of at least 3. 
 
     
     
         20 . The method of  claim 19  wherein for dissembling the fusion vector containing n vector entities into single vector entities, steps (A), (B), and (C1) are carried out for selecting an appropriate fusion vector containing 2 to (n−1) vector entities, and steps (A), (B), and (C2) to (E) are carried out with said selected fusion vector containing 2 to (n−1) vector entities. 
     
     
         21 . The method of  claim 20  wherein (n−1) of the vector entities in said fusion vector containing n vector entities each contains a further selectable marker different from the resistance markers such that only host cells transformed with fusions between a vector entity not containing the further selectable marker and one or more of the vector entities containing the selectable marker are viable in step (C1). 
     
     
         22 . The method of  claim 21  wherein (n−1) of the vector entities comprise a conditional origin of replication making the propagation of said vector entities dependent on the presence or absence of a specific gene in the host cells. 
     
     
         23 . The method of  claim 22  wherein the host cells are bacteria, preferably  E. coli , the origin of replication is R6Kγ or a derivative thereof, and the bacteria are pir 
     
     
         24 . The method of  claim 19  wherein each of the n vector entities comprises one or more genes of interest. 
     
     
         25 . The method of  claim 24  wherein the one or more genes of interest are within an expression cassette. 
     
     
         26 . A fusion vector comprising n vector entities as defined in  claim 4 , separated from each other by n of the same site-specific recombination site, wherein each vector entity contains an individual resistance marker gene different from the resistance marker genes of the other vector entities, wherein n is an integer of at least 3. 
     
     
         27 . A kit for assembly and/or disassembly of n vectors comprising
 a fusion vector comprising n vector entities each containing a multiple integration element (MIE) as defined in  claim 1 , the n vector entities being separated from each other by n of the same site-specific recombination site, wherein each vector entity contains an individual resistance marker gene different from the resistance marker genes of the other vector entities; and/or   n vector entities each containing a site-specific recombination site and an individual resistance marker gene different from the resistance marker genes of the other vector entities, and   a recombinase specific for said site-specific recombination site and/or cells for the propagation of said fusion vector and/or said n vectors vector entities;   wherein n is an integer of at least 3.