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Process for improved protein expression by strain engineering

  • US 8,603,824 B2
  • Filed: 07/26/2005
  • Issued: 12/10/2013
  • Est. Priority Date: 07/26/2004
  • Status: Active Grant
First Claim
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1. A process for engineering a recombinant bacterial cell for expression of a recombinant protein or peptide, the process comprising:

  • i) obtaining a recombinant bacterial host cell comprising a nucleic acid encoding the recombinant protein or peptide operably linked to an expression control sequence operable in said recombinant bacterial host cell;

    ii) expressing the recombinant protein or peptide in the recombinant bacterial host cell of step (i);

    iii) analyzing a genetic profile of the recombinant bacterial host cell of step (ii), wherein said genetic profile comprises at least one gene encoding a protease and at least one gene encoding a folding modulator, to identify at least one protease and at least one folding modulator that are upregulated in the recombinant bacterial host cell of step (ii) relative to a gene product of the at least one protease gene and a gene product of the at least one folding modulator gene, in either a bacterial cell that has not been modified to express the recombinant protein or peptide, or a recombinant bacterial host cell that does not express the recombinant protein or peptide, wherein the genetic profile is a transcriptome profile;

    iv) modifying the recombinant bacterial host cell of step (i) to increase the expression of the at least one upregulated folding modulator identified in step (iii) and to decrease the expression of the at least one upregulated protease identified in step (iii), by genetic modification to produce a modified recombinant bacterial host cell, wherein increasing the expression of the at least one upregulated folding modulator identified in step (iii) comprises introducing into the recombinant bacterial host cell of step (i) a DNA encoding the at least one upregulated folding modulator identified in step (iii), wherein the DNA encoding the at least one upregulated folding modulator is operably linked to an expression control sequence operable in the recombinant bacterial host cell into which it is introduced, and expressing the DNA encoding the at least one upregulated folding modulator identified in step (iii);

    v) expressing the recombinant protein or peptide in the modified recombinant bacterial host cell of step (iv);

    vi) analyzing a genetic profile of the modified recombinant bacterial host cell of step (v), wherein the genetic profile of the modified recombinant bacterial host cell of step (v) comprises at least one gene encoding a protease and at least one gene encoding a folding modulator, to identify at least one protease and at least one folding modulator that are upregulated in the modified recombinant bacterial host cell of step (v) relative to a gene product of the at least one protease gene and a gene product of the at least one folding modulator gene in a bacterial cell that has not been modified to express the recombinant protein or peptide, a recombinant bacterial host cell that does not express the recombinant protein or peptide, or a modified recombinant bacterial host cell that does not express the recombinant protein or peptide, wherein the genetic profile is a transcriptome profile;

    vii) modifying the modified recombinant bacterial host cell of step (iv) to increase the expression of the at least one upregulated folding modulator identified in step (vi) and to decrease the expression of the at least one upregulated protease identified in step (vi), in the modified recombinant host cell to produce a multiply modified recombinant bacterial host cell;

    viii) expressing the recombinant protein or peptide in the multiply modified recombinant bacterial host cell of step (vii);

    ix) measuring the amount of soluble and active recombinant protein or peptide in the multiply modified recombinant bacterial host cell of step (viii) and in the recombinant bacterial host cell of step (ii); and

    ,x) comparing the amount of soluble and active recombinant protein or peptide measured in the multiply modified recombinant bacterial host cell of step (viii) to the amount of soluble and active recombinant protein or peptide measured in the recombinant bacterial host cell of step (ii);



    whereina. the measured amount of soluble and active recombinant protein or peptide in the multiply modified recombinant bacterial host cell of step (viii) is increased relative to the measured amount of soluble and active recombinant protein or peptide in the recombinant bacterial host cell of step (ii);

    orb. the measured amount of soluble and active recombinant protein or peptide in the multiply modified recombinant bacterial host cell of step (viii) is not increased relative to the measured amount of soluble and active recombinant protein or peptide in the recombinant bacterial host cell of step (ii), and wherein steps (v) through (ix) are repeated until the amount of soluble and active recombinant protein or peptide is increased relative to the measured amount of soluble and active recombinant protein or peptide in the recombinant bacterial host cell of step (ii);

    wherein a cell comprising the increased amount of the soluble and active recombinant protein or peptide is the engineered recombinant bacterial cell.

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