Recombinant constructs and systems for secretion of proteins via type III secretion systems

A method of isolating a protein or polypeptide, said method comprising:

• providing a recombinant host cell comprising a first DNA molecule encoding a functional type III secretion system from a first source organism which is a different species or strain compared to the recombinant host cell and a second DNA molecule having a promoter operably coupled to a nucleic acid sequence encoding a protein or polypeptide secreted by the type III secretion system but from a second source organism which is a different species or strain compared to the first source organism;
• introducing the recombinant host cell into a culture medium, wherein the encoded protein or polypeptide and the type III secretion system are expressed and the encoded protein or polypeptide is secreted into the culture medium;
• and
• isolating the encoded protein or polypeptide from the culture medium.

A method of determining whether a gene encodes a potential effector protein or polypeptide comprising:

• providing a recombinant host cell comprising a DNA molecule encoding a functional type III secretion system from a source organism which is a different species or strain compared to the recombinant host cell;
• introducing into the recombinant host cell a candidate gene encoding a protein or polypeptide under conditions effective to express the encoded protein or polypeptide;
• and
• determining whether the encoded protein or polypeptide is secreted in culture by the recombinant host cell, wherein secretion of the encoded protein or polypeptide is indicative of the candidate gene encoding a potential effector protein or polypeptide.

A DNA construct consisting essentially of:

• a first DNA molecule encoding a functional type III secretion system;
• a promoter;
• and
• a second DNA molecule encoding a protein or polypeptide secreted by the encoded type III secretion system, wherein the second DNA molecule is operably coupled to said promoter so that upon introduction of the DNA construct into a host cell which does not naturally express a type III secretion system, the encoded protein or polypeptide and the encoded type III secretion system are expressed and the encoded protein or polypeptide is secreted in culture.

A method of isolating a protein or polypeptide, said method comprising:

• providing a recombinant host cell comprising a first DNA molecule encoding a functional type III secretion system from a first source organism which is a different species compared to the recombinant host cell and a second DNA molecule having a promoter operably coupled to a nucleic acid sequence encoding a protein or polypeptide secreted by the type III secretion system;
• introducing the recombinant host cell into a culture medium, wherein the encoded protein or polypeptide and the type III secretion system are expressed and the encoded protein or polypeptide is secreted into the culture medium;
• and
• isolating the encoded protein or polypeptide from the culture medium.