Dual expression vector system and screening methods

A kit comprising a vector for expressing a protein in bacterial and insect host cells, the vector comprising:

• first and second promoters, wherein the first promoter is effective to express a downstream protein coding sequence in insect host cells and the second promoter is effective to express the same downstream protein coding sequence in bacterial host cells;
• and
• a cloning site downstream of the promoters such that a protein coding sequence inserted into the site is transcribed in insect host cells and in bacterial host cells,
• wherein the second promoter is located between the first promoter and the cloning site, and
• the distance between the 3′ end of the second promoter and a ribosome binding site (RBS) is less than 100 nucleotides;
• further comprising at least one item selected from the group consisting of:
• bacterial host cells, insect host cells, baculovirus DNA, transfection reagent, agarose, an inducer, a restriction enzyme, a ligation mix, culture medium, an antibody, a buffer, a control plasmid, and instructions for use.

A method of producing a protein of interest comprising steps of:

• inserting a polynucleotide encoding a protein of interest into a cloning site of a vector for expressing a protein in bacterial and insect host cells, the vector comprising:
• first and second promoters, wherein the first promoter is effective to express a downstream protein coding sequence in insect host cells and the second promoter is effective to express the same downstream protein coding sequence in bacterial host cells;
• and
• a cloning site downstream of the promoters such that a protein coding sequence inserted into the site is transcribed in insect host cells and in bacterial host cells,
• wherein the second promoter is located between the first promoter and the cloning site, and
• the distance between the 3′ end of the second promoter and a ribosome binding site (RBS) is less than 100 nucleotides;
• introducing the resulting vector, or a recombinant baculovirus derived from the vector into a host cell;
• culturing the host cell under conditions in which the protein is expressed;
• and
• harvesting the protein.

A kit comprising a vector for expressing a protein in bacterial and insect host cells, the vector comprising:

• first and second promoters, wherein the first promoter is a baculovirus polh promoter effective to express a downstream protein coding sequence in insect host cells expressing baculovirus proteins and the second promoter is effective to express the same downstream protein coding sequence in bacterial host cells;
• and
• a cloning site downstream of the promoters such that a protein coding sequence inserted into the site is transcribed in insect host cells expressing baculovirus proteins and in bacterial host cells,
• wherein the portion of the vector between the polh promoter and the cloning site comprises at least a portion of the polh mRNA 5′ untranslated region, and wherein the portion of the polh mRNA 5′ untranslated region is not immediately followed by a portion encoding a 5′ portion of the Polh protein;
• further comprising at least one item selected from the group consisting of:
• bacterial host cells, insect host cells, baculovirus DNA, transfection reagent, agarose, an inducer, a restriction enzyme, a ligation mix, culture medium, an antibody, a buffer, a control plasmid, and instructions for use.

A method of producing a protein of interest comprising steps of:

• inserting a polynucleotide encoding a protein of interest into a cloning site of a vector for expressing a protein in bacterial and insect host cells, the vector comprising:
• first and second promoters, wherein the first promoter is a baculovirus polh promoter effective to express a downstream protein coding sequence in insect host cells expressing baculovirus proteins and the second promoter is effective to express the same downstream protein coding sequence in bacterial host cells;
• and
• a cloning site downstream of the promoters such that a protein coding sequence inserted into the site is transcribed in insect host cells expressing baculovirus proteins and in bacterial host cells,
• wherein the portion of the vector between the polh promoter and the cloning site comprises at least a portion of the polh mRNA 5′ untranslated region, and wherein the portion of the polh mRNA 5′ untranslated region is not immediately followed by a portion encoding a 5′ portion of the Polh protein;
• introducing the resulting vector, or a recombinant baculovirus derived from the vector into a host cell;
• culturing the host cell under conditions in which the protein is expressed;
• and
• harvesting the protein.