Superoxide dismutase cloning and expression in microorganisms

Abstract

Methods and compositions are provided for the production of human superoxide dismutase and a novel protocol for enhancing efficiency of expression. The gene encoding for human superoxide dismutase is isolated and inserted into a vector in conjunction with a synthetic linker which provides for enhanced efficiency in translation. E. coli strain D1210 (pSODX8) was deposited at the A.T.C.C. on Sep. 27, 1983 and given Accession No. 39453. Yeast strain 2150-2-3 (pC1/1GAPSOD) and E. coli strains D1210 (pSOD11) and D1210 (pS20R) were deposited at the A.T.C.C. on May 9, 1984, and given Accession Nos. 20708, 39679 and 39,680, respectively.

What is claimed is:  
     
         1 . A polypeptide having substantially the same amino acid sequence as human superoxide dismutase, said polypeptide having been prepared in a unicellular microorganism.  
     
     
         2 . A polypeptide according to  claim 1 , wherein said unicellular microorganism is a bacterium.  
     
     
         3 . A polypeptide according to  claim 2 , wherein said bacterium is  E. coli.    
     
     
         4 . A polypeptide according to  claim 1 , wherein said unicellular microorganism is a yeast.  
     
     
         5 . A polypeptide according to  claim 4 , wherein said yeast is  S. cerevisiae.    
     
     
         6 . A DNA construct functional in a microorganism comprising in downstream order of transcription: 
 (1) a promoter;    (2) a ribosomal binding site;    (3) an initiation codon;    (4) a human superoxide dismutase structural gene in reading frame with said initiation codon having a stop codon at the 3′-terminus; and    (5) a transcription terminator.    
     
     
         7 . A DNA construct according to  claim 6 , wherein the distance between said ribosomal binding site and the initiation codon is optimized by preparing a variety of DNA bridges between said ribosomal binding site and said initiation codon varying in length and composition, and selecting for optimized expression.  
     
     
         8 . A DNA construct according to  claim 6 , including an operator in functional relationship with said promoter.  
     
     
         9 . A DNA construct according to  claim 6 , joined to a replication system for extrachromosomal replication and maintenance.  
     
     
         10 . A DNA construct according to  claim 6 , wherein said unicellular microorganism is a bacterium.  
     
     
         11 . A DNA construct according to  claim 10 , wherein said bacterium is  E. coli.    
     
     
         12 . A DNA construct according to  claim 6 , wherein said unicellular microorganism is a yeast.  
     
     
         13 . A DNA construct according to  claim 12 , wherein said yeast is  S. cerevisiae.    
     
     
         14 . A method for enhancing translational efficiency of a gene encoding a polypeptide, in a DNA construct containing a ribosomal binding site, and an initiation codon separated by a polynucleotide spacer, said method comprising: 
 synthesizing a mixture of linkers having overhangs varying in length, including at least a portion of said spacer, and varying in composition in the region of said spacer;    joining said mixture of linkers to flanking regions to provide a DNA construct having transcriptional and translational initiation and termination regulatory signal sequences upstream and downstream, respectively from said gene and varying spacer regions between said ribosomal binding site and said initiation codon;    providing for filling in of said overhang; and    cloning said construct and screening said clones for efficiency of translation.    
     
     
         15 . A method for enhancing translational efficiency of a gene encoding a polypeptide, in a DNA construct containing a ribosomal binding site, and an initiation codon separated by a polynucleotide spacer, said method comprising: 
 synthesizing a mixture of linkers having overhangs varying in length, including at least a portion of said spacer, and varying in composition at least in the region of said spacer and from 0 to 4 codons at codon degenerate sites at the 5′-end of the coding strand;    joining said mixture of linkers to flanking regions to provide a DNA construct having transcriptional and translational initiation and termination regulatory signal sequences upstream and downstream, respectively from said gene and varying spacer regions between said ribosomal binding site and said initiation codon;    providing for filling in of said overhang; and    cloning said construct and screening said clones for efficiency of translation.    
     
     
         16 . An acetylated polypeptide having substantially the same amino acid sequence as human superoxide dismutase, said polypeptide having been prepared in yeast.  
     
     
         17 . An acetylated polypeptide as in  claim 16 , wherein the first two amino acids at the N-terminus are alanine and threonine.  
     
     
         18 . An acetylated polypeptide having substantially the same amino acid sequence as human superoxide dismutase, said polypeptide having been prepared by: 
 growing a yeast host in a suitable medium, said yeast host expressing a DNA sequence encoding the amino acid sequence of human superoxide dismutase and having an acetylation signal sequence at its 5′-end;    isolating the acetylated polypeptide from the yeast host.    
     
     
         19 . An acetylated polypeptide as in  claim 18 , wherein the acetylation signal sequence comprises the first two amino acids of the polypeptide.  
     
     
         20 . An acetylated polypeptide as in  claim 19 , wherein the first two amino acids are alanine and threonine.  
     
     
         21 . A method for producing an acetylated polypeptide, said method comprising introducing into yeast a DNA sequence encoding said polypeptide and having an acetylation signal sequence at its 5′-end, which acetylation signal sequence causes the yeast host to acetylate the N-terminal amino acid on the polypeptide.  
     
     
         22 . A method as in  claim 21 , wherein the acetylation signal sequence encodes for two amino acids at the N-terminal end of the polypeptide, said two amino acids consisting of a glycine or alanine at the first position followed by a polar amino acid at the second position.  
     
     
         23 . A method as in  claim 22 , wherein the polar amino acid is selected from the group consisting of aspartate, serine, and threonine.  
     
     
         24 . A method as in  claim 23 , wherein the two amino acids are alanine at the first position and threonine at the second position.  
     
     
         25 . An acetylated polypeptide as in  claim 18 , wherein said method of preparation includes dialysis of the polypeptide against solutions containing copper and/or zinc sulfate.  
     
     
         26 . A method for selection of yeast strains synthesizing high levels of superoxide dismutase comprising growing said yeast strains in at least 2.5 mM copper sulfate.  
     
     
         27 . A human genomic DNA sequence comprising the gene for superoxide dismutase.  
     
     
         28 . A human gene as in  claim 27  wherein said superoxide dismutase is cytoplasmic species of the enzyme.  
     
     
         29 . A human gene as in  claim 27 , wherein said superoxide dismutase is the extracellular species of the enzyme.