Secreted Proteins and Nucleic Acids Encoding Them

Abstract

The invention involves a method of identifying nucleic acid sequences encoding signal peptide-containing proteins. The method features chimeric constructs containing a KRE9 gene that lacks a signal sequence. Yeast containing chimeric KRE9 plasmid constructs that encode signal sequences are selected based on their ability to grow on media in which sucrose is the sole carbon source.

What is claimed is:  
     
         1 . A method comprising: 
 (a) obtaining a nucleic acid molecule comprising a chimeric gene, said chimeric gene comprising a first portion and a second portion, the first portion encoding a KRE9 lacking a functional signal sequence and the second portion being a heterologous nucleic acid sequence;    (b) transforming a yeast cell lacking a functional KRE9 gene with said nucleic acid molecule; and    (c) determining whether said transformed yeast cell grows when supplied with a medium that permits growth of a yeast cell expressing KRE9 having a functional signal sequence, but does not permit growth of a yeast cell that does not express KRE9 having a functional signal sequence, wherein growth on said medium indicates that said heterologous nucleic acid sequence present in said yeast cell encodes a signal sequence.    
     
     
         2 . The method of  claim 1 , wherein step (a) comprises: 
 (i) obtaining double-stranded DNA;    (ii) ligating said double-stranded DNA to a DNA molecule encoding KRE9 lacking a functional signal sequence to create a chimeric gene.    
     
     
         3 . The method of  claim 1 , wherein step (a) comprises: 
 (i) obtaining double-stranded DNA;    (ii) ligating said double-stranded DNA to a DNA molecule encoding KRE9 lacking a functional signal sequence to create a chimeric gene;    (iii) transforming a bacterium with said nucleic acid molecule comprising a chimeric gene;    (iv) growing said transformed bacterium; and    (v) isolating said nucleic acid molecule comprising a chimeric gene from said transformed bacterium.    
     
     
         4 . The method of  claim 1 , further comprising, in order to identify said signal sequence, isolating and sequencing a portion of the chimeric gene contained within a yeast cell that grows when supplied with a medium that permits growth of a yeast cell expressing KRE9, but does not permit growth of a yeast cell that does not express KRE9 having a functional signal sequence.  
     
     
         5 . The method of  claim 1 , wherein said second portion of said nucleic acid molecule is pBOSS1.  
     
     
         6 . The method of  claim 1 , wherein said second portion of said nucleic acid molecule is cDNA.  
     
     
         7 . The method of  claim 1 , wherein the yeast strain is Yscreen2.  
     
     
         8 . The method of  claim 1 , wherein said medium contains glucose as the sole carbon source.  
     
     
         9 . The method of  claim 8 , wherein the medium contains a calcineurin inhibitor.  
     
     
         10 . The method of  claim 4 , further comprising using a nucleic acid molecule encoding said signal sequence to screen an eukaryotic library for a full-length gene or cDNA encoding a protein comprising said identified signal sequence.  
     
     
         11 . A yeast cell transformed with a nucleic acid molecule comprising a chimeric gene, said chimeric gene comprising a first portion and a second portion, the first portion encoding a KRE9 lacking a functional signal sequence and the second portion being a heterologous nucleic acid sequence.  
     
     
         12 . A method comprising: 
 (a) obtaining a nucleic acid molecule comprising a chimeric gene, said chimeric gene comprising a first portion and a second portion, the first portion encoding a KRE9 lacking a functional signal sequence and the second portion being a heterologous nucleic acid sequence;    (b) transforming a yeast cell lacking a functional KRE9 gene with said nucleic acid molecule; and    (c) determining whether said transformed yeast cell grows when supplied with a medium that does not permit growth of a yeast cell expressing KRE9 having a functional signal sequence, but does permit growth of a yeast cell that does not express KRE9 having a functional signal sequence, wherein lack of growth on said medium indicates that said heterologous nucleic acid sequence present in said yeast cell encodes a signal sequence.    
     
     
         13 . The method of  claim 12 , wherein the medium contains K1 killer toxin.  
     
     
         14 . The method of  claim 12 , wherein step (a) comprises: 
 (i) obtaining a double-stranded DNA; and    (ii) ligating said double-stranded DNA to a DNA molecule encoding KRE9 lacking a functional signal sequence to create a chimeric gene.    
     
     
         15 . The method of  claim 12 , further comprising, in order to identify said signal sequence, isolating and sequencing a portion of the chimeric gene contained within said yeast cell that does not grow when supplied with a medium that does not permit growth of a yeast cell expressing KRE9, but does permit growth of a yeast cell that does not express KRE9 having a functional signal sequence.  
     
     
         16 . The expression vector pBOSS-1.  
     
     
         17 . A genetically engineered host cell comprising the vector of  claim 16.