Recombinant dna, method for preparing it and production of foreign proteins by unicellular hosts containing it

Claim 1

A method for providing for the production in a unicellular host of a protein foreign to said host, said method employing a functional recombinant DNA having a vector compatible with said host and having an intact replicon and promoter site effective for a DNA sequence for expressing said protein, said DNA sequence including a start site having a synthetically introduced deoxyribonucleotide sequence coding for a simulated robosomal binding site said method comprising joining under annealing and/or ligating conditions to form a functional recombinant DNA structure said foreign DNA sequences and a vector compatible with said host, wherein said vector and said foreign DNA sequences are separated by a synthetically introduced deoxynucleotide oligopolymer encoding a simulated ribosomal binding site recognized by said host, which oligopolymer is adjacent a deoxynucleotide triplet encoding for methionine to define a start site; and transforming said host with said recombinant DNA structure, whereby said host achieves the capability of expressing said foreign DNA sequence to produce said foreign protein.

Claim 2

A method for providing for the production in a unicellular host of a protein foreign to said host, said method employing a functional recombinant DNA having a vector compatible with said host and having an intact replicon and promoter site effective for a DNA sequence for expressing said protein, said DNA sequence including a start site having a synthetically introduced deoxyribonucleotide sequence coding for a simulated robosomal binding site said method comprising joining under annealing and/or ligating conditions to form a functional recombinant DNA structure said foreign DNA sequences and a vector compatible with said host, wherein said vector and said foreign DNA sequences are separated by a synthetically introduced deoxynucleotide oligopolymer encoding a simulated ribosomal binding site recognized by said host, which oligopolymer is adjacent a deoxynucleotide triplet encoding for methionine to define a start site; and transforming said host with said recombinant DNA structure, whereby said host achieves the capability of expressing said foreign DNA sequence to produce said foreign protein.

Claim 4

A method according to any of Claims 1 and 2, wherein said foreign DNA sequence is derived from a eukaryotic source.

Claim 6

A method according to any of Claims 1 and 2, wherein said vector is obtained by endonuclease cleavage of a plasmid to provide staggered ends.

Claim 13

A method for preparing a functional recombinant DNA structure having a DNA sequence capable of independent expression which comprises annealing and/or ligating (1) a first DNA sequence comprising a DNA sequence from a first source expressing said protein; (2) a second DNA sequence comprising a vector from a second source which does not exchange information with said first source, said vector having an intact replicon and promoter for said first DNA sequence, wherein a third DNA sequence is introduced between said first and second DNA sequences prior to or concomitantly with said annealing and/or ligating encoding for a ribosomal binding site for said second source, said third DNA sequence introduced adjacent a deoxyribonucleotide triplet encoding for methionine on said first DNA sequence, said third DNA sequence and said triplet defining a start site.

Claim 14

A method for preparing a functional recombinant DNA structure having a DNA sequence capable of independent expression which comprises annealing and/or ligating (1) a first DNA sequence comprising a DNA sequence from a first source expressing said protein; (2) a second DNA sequence comprising a vector from a second source which does not exchange information with said first source, said vector having an intact replicon and promoter for said first DNA sequence, wherein a third DNA sequence is introduced between said first and second DNA sequences prior to or concomitantly with said annealing and/or ligating encoding for a ribosomal binding site for said second source, said third DNA sequence introduced adjacent a deoxyribonucleotide triplet encoding for methionine on said first DNA sequence, said third DNA sequence and said triplet defining a start site.