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Title: Systems and methods for the secretion of recombinant proteins in gram negative bacteria

Abstract

Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

Inventors:
; ; ;
Publication Date:
Research Org.:
WISCONSIN ALUMNI RESEARCH FOUNDATION, Madison, WI (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1344502
Patent Number(s):
9,574,197
Application Number:
15/211,632
Assignee:
WISCONSIN ALUMNI RESEARCH FOUNDATION CHO
DOE Contract Number:
FC02-07ER64494
Resource Type:
Patent
Resource Relation:
Patent File Date: 2016 Jul 15
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Withers, III, Sydnor T., Dominguez, Miguel A., DeLisa, Matthew P., and Haitjema, Charles H. Systems and methods for the secretion of recombinant proteins in gram negative bacteria. United States: N. p., 2017. Web.
Withers, III, Sydnor T., Dominguez, Miguel A., DeLisa, Matthew P., & Haitjema, Charles H. Systems and methods for the secretion of recombinant proteins in gram negative bacteria. United States.
Withers, III, Sydnor T., Dominguez, Miguel A., DeLisa, Matthew P., and Haitjema, Charles H. Tue . "Systems and methods for the secretion of recombinant proteins in gram negative bacteria". United States. doi:. https://www.osti.gov/servlets/purl/1344502.
@article{osti_1344502,
title = {Systems and methods for the secretion of recombinant proteins in gram negative bacteria},
author = {Withers, III, Sydnor T. and Dominguez, Miguel A. and DeLisa, Matthew P. and Haitjema, Charles H.},
abstractNote = {Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Tue Feb 21 00:00:00 EST 2017},
month = {Tue Feb 21 00:00:00 EST 2017}
}

Patent:

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  • Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.
  • The authors report the overexpression, purification, and properties of the regulatory protein, MerR, for a chromosomally encoded mercury resistance determinant from Bacillus strain RC607. This protein is similar in sequence to the metalloregulatory proteins encoded by gram-negative resistance determinants found on transposons Tn21 and Tn501 and to a predicted gene product of a Staphylococcus aureus resistance determinant. In vitro DNA-binding and transcription experiments were used to demonstrate those purified Bacillus MerR protein controls transcription from a promoter-operator site similar in sequence to that found in the transposon resistance determinants. The Bacillus MerR protein bound in vitro to its promoter-operator regionmore » in both the presence and absence of mercuric ion and functioned as a negative and positive regulator of transcription. The MerR protein bound less tightly to its operator region (ca. 50- to 100-fold) in the presence of mercuric ion; this reduced affinity was largely accounted for by an increased rate of dissociation of the MerR protein from the DNA. Despite this reduced DNA-binding affinity, genetic and biochemical evidence support a model in which the MerR protein-mercuric ion complex is a positive regulator of operon transcription. Although the Bacillus MerR protein bound only weakly to the heterologous Tn501 operator region, the Tn501 and Tn21 MerR proteins bound with high affinity to the Bacillus promoter-operator region and exhibited negative, but not positive, transcriptional control.« less
  • Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.
  • The present invention provides a method of targeted mutagenesis in Gram-positive bacteria. In particular, the present invention provides a method that effectively integrates a suicide integrative vector into a target gene in the chromosome of a Gram-positive bacterium, resulting in inactivation of the target gene.
  • The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ionsmore » such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.« less