Vector system for selection of genes encoding secreted proteins and membrane-bound proteins

Abstract

The subject invention concerns novel vectors for the rapid and robust selection for cDNA sequences that encode secreted or membrane-bound proteins. The invention also pertains to methods for cloning secreted or membrane-bound proteins, including proteins encoded by novel members of gene families.

1 . A method for selecting nucleic acid sequences encoding secreted or membrane-bound proteins which comprises: 
 (a) linearizing a vector with one or more restriction enzymes, wherein the vector comprises DNA encoding a reporter molecule lacking a functional signal sequence;    (b) cutting a candidate nucleic acid sequence with the one or more restriction enzymes and ligating the candidate nucleic acid sequence to the linearized vector, thereby forming a ligation product, wherein the candidate nucleic acid sequence encodes a potential secreted or membrane-bound protein;    (c) transforming bacterial cells with the ligation product; and    (d) selecting for colonies based on expression of the reporter gene functionally linked to the secreted or membrane-bound protein.    
     
     
         2 . The method according  claim 1 , further comprising the steps of (e) determining the nucleic acid sequence within the transformants from the selected colonies and (f) determining the amino acid sequence based on the nucleic acid sequence.  
     
     
         3 . The method according to  claim 1 , wherein the reporter molecule is β-lactamase or alkaline phosphatase.  
     
     
         4 . The method according  claim 1 , wherein the nucleic acid sequence of the vector is SEQ ID NO. 1 or SEQ ID NO. 2.  
     
     
         5 . A method for selecting nucleic acid sequences encoding proteins that are secreted or membrane-bound which comprises: 
 (a) linearizing a vector with one or more restriction enzymes, wherein the vector comprises DNA encoding β-lactamase lacking a functional signal sequence;    (b) cutting a candidate nucleic acid sequence with the one or more restriction enzymes and ligating the candidate nucleic acid to the linearized vector, thereby forming a ligation product, wherein the candidate nucleic acid sequence encodes proteins that are potentially secreted or membrane-bound;    (c) transforming bacterial cells with the ligation product; and    (d) selecting for colonies based on selection criteria.    
     
     
         6 . The method according to  claim 5 , further comprising the steps of e) determining the nucleic acid sequence within the transformants of the selected colonies and f) determining the amino acid sequence based on the nucleic acid sequence.  
     
     
         7 . The method according to  claim 5 , wherein the nucleic acid sequence of the linearized vector comprises SEQ ID NO. 1 or SEQ ID NO. 2.  
     
     
         8 . A vector for selection of nucleic acid sequences encoding proteins that are secreted or membrane-bound comprising a reporter gene, wherein said reporter gene encodes a reporter molecule lacking a functional signal sequence; a selectable marker gene, wherein said reporter gene and said selectable marker gene are operably linked to a promoter sequence; and a multiple cloning site.  
     
     
         9 . The vector of  claim 8 , wherein said reporter gene is β-lactamase and said promoter sequence is a lac promoter sequence.  
     
     
         10 . The vector of  claim 8 , wherein said reporter gene is operably linked to a first promoter sequence and said selectable marker gene is operably linked to a second promoter sequence.  
     
     
         11 . The vector of  claim 8 , wherein said reporter gene is alkaline phosphatase.  
     
     
         12 . The vector of  claim 8 , wherein said selectable marker gene is selected from the group consisting of a neomycin-resistance gene, tetracycline-resistance gene, chloramphenicol-resistance gene, and bleomycin-resistance gene.  
     
     
         13 . The vector of  claim 8 , wherein said vector comprises the nucleic acid sequence of SEQ ID NO. 1.  
     
     
         14 . A vector for selection of nucleic acid sequences encoding proteins that are secreted or membrane-bound comprising a reporter gene, wherein said reporter gene encodes a reporter molecule lacking a functional signal sequence; a selectable marker gene, wherein said reporter gene and said selectable marker gene are operably linked to a promoter sequence; a nucleotide sequence comprising a plurality of thymidine nucleotides; and a multiple cloning site.  
     
     
         15 . The vector of  claim 14 , wherein said reporter gene is the β-lactamase gene and said promoter sequence is a lac promoter sequence.  
     
     
         16 . The vector of  claim 14 , wherein said reporter gene is operably linked to a first promoter sequence and said selectable marker gene is operably linked to a second promoter sequence.  
     
     
         17 . The vector of  claim 14 , wherein said reporter gene is the alkaline phosphatase gene.  
     
     
         18 . The vector of  claim 14 , wherein said selectable marker gene is selected from the group consisting of a neomycin-resistance gene, tetracycline-resistance gene, chloramphenicol-resistance gene, and bleomycin-resistance gene.  
     
     
         19 . The vector of  claim 14 , wherein said sequence comprising a plurality of thymidine nucleotides allows transcriptional or translational slippage.  
     
     
         20 . The vector of  claim 14 , wherein said sequence comprising a plurality of thymidines comprises about 10 thymidine nucleotides to about 30 thymidine nucleotides.  
     
     
         21 . The vector of  claim 14 , wherein said sequence comprising a plurality of thymidine nucleotides comprises about 10 thymidine nucleotides to about 20 thymidine nucleotides.  
     
     
         22 . The vector of  claim 14 , wherein said sequence comprising a plurality of thymidine nucleotides comprises 13 thymidine nucleotides.  
     
     
         23 . The vector of  claim 14 , wherein said vector comprises the nucleic acid sequence of SEQ ID NO. 2.  
     
     
         24 . A method for selecting nucleic acid sequences encoding secreted or membrane-bound proteins which comprises: 
 (a) providing a linearized vector comprising a reporter gene, wherein said reporter gene encodes a reporter molecule lacking a functional signal sequence; a selectable marker gene, wherein said reporter gene and said selectable marker gene are operably linked to a promoter sequence; and a multiple cloning site;    (b) ligating a candidate nucleic acid sequence to the linearized vector, thereby forming a ligation product, wherein the candidate nucleic acid sequence encodes a potential secreted or membrane-bound protein;    (c) transforming bacterial cells with the ligation product; and    (d) selecting for colonies based on expression of the selectable marker gene and the reporter gene functionally linked to the secreted or membrane-bound protein.    
     
     
         25 . The method according to  claim 24 , further comprising the steps of (e) determining the nucleic acid sequence within the transformants from the selected colonies and (f) determining the amino acid sequence based on the nucleic acid sequence.  
     
     
         26 . The method according to  claim 24 , wherein the reporter gene is the β-lactamase gene and the promoter sequence is a lac promoter sequence.  
     
     
         27 . The method according to  claim 24 , wherein the reporter gene is operably linked to a first promoter sequence and the selectable marker gene is operably linked to a second promoter sequence.  
     
     
         28 . The method of  claim 24 , wherein the reporter gene is the alkaline phosphatase gene.  
     
     
         29 . The method of  claim 24 , wherein the selectable marker gene is selected from the group consisting of the neomycin-resistance gene, tetracycline-resistance gene, chloramphenicol-resistance gene, and bleomycin-resistance gene.  
     
     
         30 . The method according to  claim 24 , wherein the linearized vector further comprises a plurality of thymidine nucleotides.  
     
     
         31 . The method according to  claim 24 , wherein the linearized vector comprises the nucleic acid sequence of SEQ ID NO. 1 or SEQ ID NO:2.