Hybrid prokaryotic polypeptides produced by in vivo homologous recombination

An enzymatically active hybrid procaryotic amylase encoded by a hybrid DNA sequence, said hybrid DNA sequence being produced by a process comprising;

• forming a circular vector comprising a replicable DNA sequence, a first DNA sequence encoding at least one amino acid of the amino-terminal portion of said hybrid amylase, a second DNA sequence encoding at least one amino acid of the carboxy-terminal portion of said hybrid amylase and a third DNA sequence between said first and second DNA sequences;
• transforming a rec positive microorganism with said vector to produce a cell population containing a multiplicity of said circular vector and permitting crossover recombination within at least one of said vectors to excise said third DNA sequence to form a recombined circular vector having a hybrid DNA sequence comprising said first and said second DNA sequences encoding said enzymatically active hybrid amylase;
• and,
• and detecting expression of said hybrid DNA sequence by assaying for amylase activity.

An enzymatically active hybrid procaryotic amylase encoded by a hybrid DNA sequence, said hybrid DNA sequence being produced by a process comprising;

• forming a circular vector comprising a replicable DNA sequence, a first DNA sequence comprising a functional promoter operably linked to sequences encoding at least one amino acid of the amino-terminal portion of said hybrid amylase, a second DNA sequence encoding at least one amino acid of the carboxy-terminal portion of said hybrid amylase and a third DNA sequence between said first and said second DNA sequences, said third DNA sequence comprising a DNA sequence which prevents the expression of said second DNA sequence;
• transforming a rec portion microorganism with said circular vector to produce a cell population containing a multiplicity of said circular vector and permitting crossover recombination within at least one or said circular vectors to excise said third DNA sequence to form a recombined circular vector having a hybrid DNA sequence comprising said first and said second DNA sequences encoding said enzymatically active hybrid amylase;
• and
• detecting the expression of said hybrid DNA sequence by assaying for amylase activity.

An enzymatically active hybrid procaryotic amylase encoded by a hybrid DNA sequence, said hybrid DNA sequence being produced by a process comprising;

• forming a circular vector comprising replicable DNA sequence, a first DNA sequence comprising a functional promoter operably linked to sequences encoding at least one amino acid of the amino-terminal portion of said hybrid amylase, a second DNA sequence encoding at least one amino acid of the carboxy-terminal portion of said hybrid amylase, a third DNA sequence between said first and said second DNA sequences, said third DNA sequence comprising a DNA sequence which prevents the expression of said second DNA sequence and a fourth DNA sequence 3' to said second DNA sequence, said fourth DNA sequence encoding a defined selection characteristic;
• transforming a rec positive microorganism with said circular vector to produce a cell population containing a multiplicity of said circular vector and permitting crossover recombination within at least one of said circular vectors to excise said third DNA sequence to form a recombined circular vector having a hybrid DNA sequence comprising said first, said second and said fourth DNA sequences encoding said enzymatically active hybrid amylase and said defined selection characteristic;
• and
• selecting transformants which express said defined selection characteristic of said fourth DNA sequence
• and detecting expression of said hybrid DNA sequence by assaying for amylase activity.

An enzymatically active hybrid procaryotic amylase encoded by a hybrid DNA sequence, said hybrid DNA sequence being produced by a process comprising;

• forming a circular vector comprising a replicable DNA sequence, a first DNA sequence comprising a functional promoter operably linked to a sequence encoding at least one amino acid of the amino-terminal portion of said hybrid amylase corresponding to a first part of a first parental amylase sequence, a second DNA sequence encoding at least one amino acid of the carboxy-terminal portion of said hybrid amylase corresponding to a first part of a second parental amylase sequence and a third DNA sequence between said first and said second DNA sequence encoding a second part of said first parental amylase sequence and a second part of said second parental amylase sequence;
• transforming a rec positive microorganism with said circular vector to produce a cell population containing a multiplicity of said circular vector rand permitting crossover recombination within at lest one of said circular vectors to excise said hybrid DNA sequence to form a recombined circular vector having a hybrid DNA sequence comprising said first and said second DNA sequences encoding said enzymatically active hybrid amylase;
• and
• identifying transformants containing said recombined circular vector by detecting expression of said hybrid DNA sequence by assaying for amylase activity.

An enzymatically active hybrid procaryotic protease encoded by a hybrid DNA sequence, said hybrid DNA sequence being produced by a process comprising;

• forming a circular vector comprising a replicable DNA sequence, a first DNA sequence encoding at least one amino acid of the amino-terminal portion of said hybrid protease, a second DNA sequence encoding at least one amino acid of the carboxy-terminal portion of said hybrid protease and a third DNA sequence between said first and second DNA sequences;
• transforming a rec positive microorganism with said vector to produce a cell population containing a multiplicity of said circular vector and permitting crossover recombination within at least one of said vectors to excise said third DNA sequence to form a recombined circular vector having a hybrid DNA sequence comprising said firs and said second DNA sequences encoding said enzymatically active hybrid protease;
• and,
• and detecting expression of said hybrid DNA sequence by assaying for protease activity.

An enzymatically active hybrid procaryotic protease encoded by a hybrid DNA sequence, said hybrid DNA sequence being produced by a process comprising;

• forming a circular vector comprising a replicable DNA sequence, a first DNA sequence comprising a functional promoter operably linked to sequences encoding at least one amino acid of the amino-terminal portion of said hybrid protease, a second DNA sequence encoding at least one amino acid of the carboxy-terminal portion of said hybrid protease and a third DNA sequence between said first and said second DNA sequences, said third DNA sequence comprising a DNA sequence which prevents the expression of said second DNA sequence;
• transforming a rec positive microorganism with said circular vector to produce a cell population containing a multiplicity of said circular vector and permitting crossover recombination within at least one or said circular vectors to excise said third DNA sequence to form a recombined circular vector having a hybrid DNA sequence comprising said first and said second DNA sequences encoding said enzymatically active hybrid protease;
• and
• detecting the expression of said hybrid DNA sequence by assaying for protease activity.

An enzymatically active hybrid procaryotic protease encoded by a hybrid DNA sequence, said hybrid DNA sequence being produced by a process comprising;

• forming a circular vector comprising a replicable DNA sequence, a first DNA sequence comprising a functional promoter operably linked to sequences encoding at least one amino acid of the amino-terminal portion of said hybrid protease, a second DNA sequence encoding at least one amino acid of the carboxy-terminal portion of said hybrid protease, a third DNA sequence between said first and said second DNA sequences, said third DNA sequence comprising a DNA sequence which prevents the expression of said second DNA sequence and a fourth DNA sequence 3' to said second DNA sequence, said fourth DNA sequence encoding a defined selection characteristic;
• transforming a rec positive microorganism with said circular vector to produce a cell population containing a multiplicity of said circular vector and permitting crossover recombination within at least one of said circular vectors to excise said third DNA sequence to form a recombined circular vector having a hybrid DNA sequence comprising said first, said second and said fourth DNA sequences encoding said enzymatically active hybrid protease and said defined selection characteristic;
• and
• selecting transformants which express said defined selection characteristic of said fourth DNA sequence
• and detecting expression of said hybrid DNA sequence by assaying for protease activity.

An enzymatically active hybrid procaryotic protease encoded by a hybrid DNA sequence, said hybrid DNA sequence being produced by a process comprising;

• forming a circular vector comprising a replicable DNA sequence, a first DNA sequence comprising a functional promoter operably linked to a sequence encoding at least one amino acid of the amino-terminal portion of said hybrid protease corresponding to a first part of a first parental protease sequence, a second DNA sequence encoding at least one amino acid of the carboxy-terminal portion of said hybrid protease corresponding to a first part of a second parental protease sequence and a third DNA sequence between said first and said second DNA sequence encoding a second part of said first parental protease sequence and a second part of said second parental protease sequence;
• transforming a rec positive microorganism with said circular vector to produce a cell population containing a multiplicity of said circular vector and permitting crossover recombination within at lest one of said circular vectors to excise said third DNA sequence to form a recombined circular vector having a hybrid DNA sequence comprising said first and said second DNA sequences encoding said enzymatically active hybrid protease;
• and
• identifying transformants containing said recombined circular vector by detecting expression of said hybrid DNA sequence by assaying for protease activity.