Protein expression systems

a protein and expression system technology, applied in the field of protein expression systems, can solve the problems of reducing the fermentation efficiency of the production of the desired recombinant, unable to survive host cells lacking the selection marker, such as revertants, and unable to achieve the production of superior host cells. , to achieve the effect of improving the production of bacteria's protein

Inactive Publication Date: 2005-08-25
PFENEX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0020] It has been discovered that bacterial protein production can be improved by selecting as a host cell a Pseudomonad organism t

Problems solved by technology

There are many hurdles to the creation of a superior host cell.
Such revertants can decrease the fermentation efficiency of the production of the desired recombinant polypeptide.
Host cells lacking the selection marker, such as revertants, are unable to survive.
The use of selection markers during the fermentation process is intended to ensure that only bacteria containing the expression vector survive, eliminating competition between the revertants and transformants and reducing the efficiency of fermentation.
The presence of antibiotic resistance genes in a bacterial host cell, however, presents environmental, regulatory, and commercial problems.
For example, antibiotic resistance gene-containing products (and products produced by the use of antibiotic resistance gene) have been identified as potential biosafety risks for environmental, human, and animal health.
Clearance of these agents, and especially demonstrating such clearance, is expensive, time consuming, and often only minimally effective.
For example, auxotrophic markers have been widely utilized in yeast, due largely to the inefficiency of antibiotic resistance selection markers in these host cells.
However, yeast expression systems due not provide the potential speed and efficiency for producing target pr

Method used

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  • Protein expression systems
  • Protein expression systems
  • Protein expression systems

Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of a pyrF Selection Marker System in a P. fluorescens Host Cell Expression System

[0279] Reagents were acquired from Sigma-Aldrich (St. Louis Mo.) unless otherwise noted. LB is 10 g / L tryptone, 5 g / L yeast extract and 5 g / L NaCI in a gelatin capsule (BIO 101). When required, uracil (from BIO101, Carlsbad Calif.) or L-proline was added to a final concentration of 250 ug / mL, and tetracycline was added to 15 ug / mL. LB / 5-FOA plates contain LB with 250 mM uracil and 0.5 mg / mL 5-fluoroorotic acid (5-FOA). M9 media consists of 6 g / L Na2HPO4, 3 g / L KH2PO4, 1 g / L NH4Cl, 0.5 g / L NaCl, 10 mM MgSO4, 1× HoLe Trace Element Solution, pH7. Glucose was added to a final concentration of 1%. The 1000× HoLe Trace Element Solution is 2.85 g / L H3BO3, 1.8 g / L MnCl2 . 4H2O, 1.77 g / L sodium tartrate, 1.36 g / L FeSO4. 7H2O, 0.04 g / L CoCl2. 6H2O, 0.027 g / L CuCl2. 2H2O, 0.025 g / L Na2MoO4. 2H2O, 0.02 g / L ZnCl2.

Oligonucleotides Used Herein

[0280]

MB214pyrF1 (NotI site in bold)5′-GCGGCCGCTTTGGCGCTTCGT...

example 2

Construction of a pyrF—proC Dual Auxotrophic Selection Marker System in a P. fluorescens Host Cell Expression System

[0292] Oligonucleotides Used Herein

proC15′-ATATGAGCTCCGACCTTGAGTCGGCCATTG-(SEQ ID NO: 22)3′proC25′-ATATGAGCTCGGATCCAGTACGATCAGCAGG(SEQ ID NO: 23)TACAG-3′proC35′-AGCAACACGCGTATTGCCTT-3′(SEQ ID NO: 24)proC55′-GCCCTTGAGTTGGCACTTCATCG-3′(SEQ ID NO: 25)proC65′-GATAAACGCGAAGATCGGCGAGATA-3′(SEQ ID NO: 26)proC75′-CCGAGCATGTTTGATTAGACAGGTCCTTATT(SEQ ID NO: 27)TCGA-3′proC85′-TGCAACGTGACGCAAGCAGCATCCA-3′(SEQ ID NO: 28)proC95′-GGAACGATCAGCACAAGCCATGCTA-3′(SEQ ID NO: 29)genF25′-ATATGAGCTCTGCCGTGATCGAAATCCAGA-(SEQ ID NO: 30)3′genR25′-ATATGGATCCCGGCGTTGTGACAATTTACC-(SEQ ID NO: 31)3′XbaNotDraU2 linker5′-TCTAGAGCGGCCGCGTT-3′(SEQ ID NO: 32)XbaNotDraL linker5′-GCGGCCGCTCTAGAAAC-3′(SEQ ID NO: 33)

Cloning of proC from P. fluorescens and Formation of a pCN Expression Plasmid Containing proC

Replacing Antibiotic Resistant Gene in pCN51lacI with proC

[0293] The proC ORF and about 100 bp of...

example 3

Chromosomal Integration of lacI. lacIQ and lacIQ1 in P. fluorescens

[0316] Three P. fluorescens strains have been constructed, each with one of three different Escherichia coli lacI alleles, lacI (SEQ ID NO:9), lacIQ (SEQ ID NO: 11), and lacIQ1 (SEQ ID NO:12), integrated into the chromosome. The three strains exhibit differing amounts of LacI repressor accumulation. Each strain carries a single copy of its lacI gene at the levansucrase locus (SEQ ID NO:13) of P. fluorescens DC36, which is an MB101 derivative (see TD Landry et al., “Safety evaluation of an α-amylase enzyme preparation derived from the archaeal order Thermococcales as expressed in Pseudomonas fluorescens biovar I,”Regulatory Toxicology and Pharmacology 37(1): 149-168(2003)) formed by deleting the pyrF gene thereof, as described above.

[0317] No vector or other foreign DNA sequences remain in the strains. The strains are antibiotic-resistance-gene free and also contain a pyrF deletion, permitting maintenance, during gr...

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Abstract

The present invention provides an improved expression system for the production of recombinant polypeptides utilizing auxotrophic selectable markers. In addition, the present invention provides improved recombinant protein production in host cells through the improved regulation of expression.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional patent application Ser. No. 60 / 523,420 filed Nov. 19, 2003, entitled “Improved Pseudomonas Expression Systems with Auxotrophic Selection Markers,” and U.S. Provisional patent application 60 / 537,147 filed Jan. 16, 2004, and entitled “Bacterial Expression Systems with Improved Repression.”FIELD OF THE INVENTION [0002] The present invention provides an improved expression system for the production of recombinant polypeptides utilizing auxotrophic selectable markers. In addition, the present invention provides improved recombinant protein production in host cells through the improved regulation of expression. BACKGROUND OF THE INVENTION [0003] The use of bacterial cells to produce protein based therapeutics is increasing in commercial importance. One of the goals in developing a bacterial expression system is the production of high quality target polypeptides quickly, efficiently, and abu...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/06C12N9/88C12N15/52C12N15/74C12N15/78C12P13/22C12P21/00C12P21/02
CPCC12N9/0028C12N9/88C12P21/02C12N15/78C12P21/00C12N15/52C12Y305/05001C12N1/20
Inventor SCHNEIDER, JANECHEW, LAWRENCEBADGLEY, ANNERAMSEIER, THOMAS
Owner PFENEX
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