Expression of maize codon optimized proteins in pseudomonas fluorescens
View Patent ↗Compositions and methods for improving expression of a recombinant protein or polypeptide of interest in a host cell are provided. Compositions comprising a polynucleotide coding sequence for a BTBooster are provided using a plant optimized system. The coding sequences can be used in vector constructs or expression systems for transformation and expression of a recombinant protein or polypeptide of interest in a host cell. Methods comprising the codon optimization of a polynucleotide coding sequence for increased expression in a bacterial host are provided. The codon optimization method can be used in designing a polynucleotide coding sequence which expresses robust levels of protein in a bacterial host cell.
1. A method for codon optimizing a polynucleotide for the expression of a protein in a bacterial host cell, the method comprising replacing at least one codon in the polynucleotide with a codon that is used more frequently in the genes of a plant than in the genes of the bacterial host cell, such that
GC and TA doublets are removed from the polynucleotide sequence;
predicted ribonucleic acid (RNA) stem-loop-forming structures are removed from the polynucleotide sequence;
at least one restriction enzyme binding sequence is removed from the polynucleotide sequence;
sequence blocks that have more than about six consecutive residues of [G+C] or [A+T] are removed from the polynucleotide sequence; and
exon:intron junctions, poly-A addition signals, and RNA polymerase termination signals are removed from the polynucleotide sequence.
2. A bacterial host cell comprising the polynucleotide produced by the method according to claim 1 .
3. The bacterial host cell of claim 2 , wherein the bacterial host cell is a Pseudomonas host cell, and Escherichia host cell, an Acidovorax host cell, and Brevundimonas host cell, an Burkholderia host cell, an Hydrogenophaga host cell, an Oceanimonas host cell, an Ralstonia host cell, an Stenotrophomonas host cell, an Sphingomonas host cell, an Xanthomonas host cell, or an Acidomonas host cell.
4. The method according to claim 1 , wherein the at least one codon is replaced with a codon that is used more frequently in the genes of a monocotyledonous plant than in the genes of the bacterial host cell, or the at least one codon is replaced with a codon that is used more frequently in the genes of a dicotyledonous plant than in the genes of the bacterial host cell.
5. The method according to claim 1 , wherein the at least one codon is replaced with a codon that is used more frequently in the genes of Zea mays than in the genes of the bacterial host cell.
6. The method according to claim 1 , wherein the bacterial host cell is a Pseudomonas host cell, and Escherichia host cell, an Acidovorax host cell, and Brevundimonas host cell, an Burkholderia host cell, an Hydrogenophaga host cell, an Oceanimonas host cell, an Ralstonia host cell, an Stenotrophomonas host cell, an Sphingomonas host cell, an Xanthomonas host cell, or an Acidomonas host cell.
7. The method according to claim 1 , wherein the bacterial host cell is a Pseudomonas fluorescens host cell.
8. The method according to claim 5 , wherein the bacterial host cell is a Pseudomonas fluorescens host cell.
9. A method for codon optimizing a polynucleotide encoding a protein for the expression of the protein in a bacterial host cell, the method comprising replacing at least one codon in the polynucleotide with a codon that is used more frequently in the genes of a plant than in the genes of the bacterial host cell.
10. The method according to claim 9 , wherein the at least one codon is replaced with a codon that is used more frequently in the genes of Zea mays than in the genes of the bacterial host cell.
11. The method according to claim 9 , wherein the bacterial host cell is a Pseudomonas fluorescens host cell.
12. The method according to claim 10 , wherein the bacterial host cell is a Pseudomonas fluorescens host cell.