Proteolytic inactivation of select proteins in bacterial extracts for improved expression

Patent metadata

Field	Value
Publication number	US-11261219-B2
Application number	US-201916562837-A
Country	US
Kind code	B2
Filing date	Sep 6, 2019
Priority date	Oct 12, 2012
Publication date	Mar 1, 2022
Grant date	Mar 1, 2022

Abstract

The present disclosure provides modified proteins that are capable of being cleaved by the protease OmpT1. The proteins can be modified in an exposed surface motif to incorporate OmpT1 cleavage sites. Also provided are nucleic acids encoding the modified proteins, bacterial cells that express the modified proteins, and cell free synthesis systems containing modified RF1. The disclosure further provides methods for reducing the deleterious activity of a modified protein in a cell free synthesis system by contacting the modified protein with OmpT1. Also provided are methods for reducing RF1 competition at an amber codon in the cell free synthesis system, and methods for expressing a protein in the cell free synthesis system. The modified proteins of the invention can be used to increase the yield of proteins having non-natural amino acids incorporated at an amber codon.

First claim

What is claimed is:

1. A bacterial cell expressing both an Outer Membrane Protein T1 (OmpT1) and a modified Releasing Factor 1 (RF1) protein, wherein the modified RF1 protein comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1, wherein the modified RF1 protein has activity to recognize a stop codon in an mRNA sequence and terminate translation, wherein the amino acid sequence of the modified RF1 comprises a scissile OmpT1 peptide bond within the amino acid sequence corresponding to amino acids 287-304 of SEQ ID NO:1, wherein the scissile OmpT1 peptide bond comprises two adjacent basic amino acids that are positively charged at pH 7.0, and the amino acid sequence of the OmpT1 peptide bond comprises:
an arginine (R) at the amino acids corresponding to positions 295 and 296 of SEQ ID NO:1;
an R or lysine (K) at the amino acids corresponding to positions 297 and 298 of SEQ ID NO:1;
an R or K at the amino acids corresponding to positions 296 and 297 of SEQ ID NO:1; or
an R or K at the amino acids corresponding to positions 296, 297, and 298 of SEQ ID NO:1;
and wherein the scissile OmpT1 bond of the modified RF1 is cleaved by the OmpT1 expressed by the bacterial cell.

2. The bacterial cell of claim 1 , wherein the bacterial cell is an Escherichia coli.

3. A method for reducing deleterious activity of a modified RF1 protein in an in vitro cell free synthesis system, the method comprising the steps of:
i) culturing an OmpT1 positive bacteria expressing the modified RF1 protein, wherein said modified RF1 protein comprises a scissile OmpT1 peptide bond comprising two adjacent basic amino acids that are positively charged at pH 7.0, wherein the scissile OmpT1 peptide bond is located within the amino acid sequence corresponding to amino acids 287-304 of SEQ ID NO: 1, where the modified RF1 protein comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1, and has activity to recognize a stop codon in an mRNA sequence and terminate translation, and the amino acid sequence of the modified RF1 comprises:
an arginine (R) at the amino acids corresponding to positions 295 and 296 of SEQ ID NO:1;
an R or lysine (K) at the amino acids corresponding to positions 297 and 298 of SEQ ID NO:1;
an R or K at the amino acids corresponding to positions 296 and 297 of SEQ ID NO: 1; or
an R or K at the amino acids corresponding to positions 296, 297, and 298 of SEQ ID NO: 1;

ii) lysing the bacteria to create a cell free synthesis extract;
iii) contacting the modified RF1 protein with wild-type OmpT1 in an amount sufficient to reduce intact modified RF1 protein by 50%;
iv) adding a nucleic acid template to the extract to produce a cell-free synthesis system, where the template encodes a protein of interest; and,
v) allowing the cell-free synthesis system to produce the protein of interest.

4. The method of claim 3 , wherein the OmpT1 positive bacteria is Escherichia coli.

5. The method of claim 3 , wherein the oxidative phosphorylation of the bacteria remains active after cell lysis and during the synthesis of the protein of interest.

6. The method of claim 3 , wherein the cell free synthesis system places a non-native amino acid at an amber codon of the protein of interest.

7. A method for reducing RF1 competition at an amber codon in an in vitro cell free synthesis system, the method comprising the steps of:
i) culturing an OmpT1 positive bacteria expressing a mutant RF1 protein that is cleavable by an OmpT1;
ii) lysing the bacteria to create a cell free synthesis extract;
iii) contacting the mutant RF1 protein in the extract with OmpT1 in an amount sufficient to reduce intact mutant RF1 protein by 50%;
iv) adding a nucleic acid template to the extract to produce a cell-free synthesis system, where the template encodes a protein of interest and includes an amber codon; and,
v) allowing the cell free synthesis system to produce the protein of interest,
where the mutant RF1 protein has a scissile OmpT1 peptide bond located within the switch loop region corresponding to amino acids 287-304 of SEQ ID NO: 1, and where the switch loop region is modified to include two adjacent basic amino acids that are positively charged at pH 7.0, wherein the mutant RF1 protein comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1 and has activity to recognize a stop codon in an mRNA sequence and terminate translation, and the amino acid sequence of the mutant RF1 protein comprises:
an arginine (R) at the amino acids corresponding to positions 295 and 296 of SEQ ID NO: 1;
an R or lysine (K) at the amino acids corresponding to positions 297 and 298 of SEQ ID NO: 1;
an R or K at the amino acids corresponding to positions 296 and 297 of SEQ ID NO: 1; or
an R or K at the amino acids corresponding to positions 296, 297, and 298 of SEQ ID NO: 1.

8. The method of claim 7 , wherein the OmpT1 positive bacteria is Escherichia coli.

9. The method of claim 7 , wherein oxidative phosphorylation of the bacteria remains active after cell lysis and during the synthesis of the protein of interest.

10. The method of claim 7 , wherein the cell free synthesis system places a non-native amino acid at an amber codon of the protein of interest.

11. The method of claim 3 , wherein the scissile OmptT1 peptide bond comprises an amino acid sequence selected from the group consisting of:

(SEQ ID NO: 6)

QQAEASTRRRLLGSGDRS,

(SEQ ID NO: 12)

QQAEASTRRKKLGSGDRS,

(SEQ ID NO: 13)

QQAEASTRRKRLGSGDRS,

(SEQ ID NO: 14)

QQAEASTRRKVLGSGDRS,

(SEQ ID NO: 15)

QQAEASTRRRKLGSGDRS,

(SEQ ID NO: 16)

QQAEASTRRRRLGSGDRS,

(SEQ ID NO: 17)

QQAEASTRRRVLGSGDRS,

(SEQ ID NO: 18)

QQAEASTRRKKKGSGDRS,

(SEQ ID NO: 19)

QQAEASTRRKKRGSGDRS,

(SEQ ID NO: 20)

QQAEASTRRKRKGSGDRS,

(SEQ ID NO: 21)

QQAEASTRRKRRGSGDRS,

Assignees

• Sutro Biopharma Inc

Inventors

• Thanos Christopher D
• Murray Christopher J
• Yang Junhao
• Stephenson Heather

Classifications

• C12Y304/21

  Serine endopeptidases (3.4.21) · CPC title

• C12P21/00

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• G01N30/06

  Preparation · CPC title

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title