Pfenex Inc.

Dow Global Technolgoies LLC

Charles H. Squires

Diane M. Retallack

David C. Watkins

Frank H. Gaertner

Robert Shutter

Stacey Lynn Lee

Charles H Squires

Diane M Retallack

Thomas Martin Ramseier

Russell J Coleman

David K Watkins

Jane C Schneider

Frank H Gaertner

Charles D Hershberger

Lawrence C Chew

Hongfan Jin

David C Watkins

Thomas M Ramseier

The present invention provides an array for rapidly identifying a host cell population capable of producing a heterologous recombinant protein with improved yield and/or quality. The array comprises one or more host cell populations that have been genetically modified to increase the expression of one or more target genes involved in protein production, decrease the expression of one or more target genes involved in protein degradation, or both. One or more of the strains in the array may express the heterologous recombinant protein of interest in a periplasm compartment or may secrete the heterologous recombinant protein extracellularly through an outer cell wall. The strain arrays are useful for screening for improved expression of any protein of interest including therapeutic proteins, hormones, growth factors, extracellular receptors or ligands, proteases, kinases, blood proteins, chemokines, cytokines, antibodies and the like.

Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins

The invention is a process for improved production of a recombinant mammalian protein by expression in a Pseudomonad, particularly in aorganism. The process improves production of mammalian proteins, particularly human or human-derived proteins, over known expression systems such asin comparable circumstances. Processes for improved production of isolated mammalian, particularly human, proteins are provided.

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Provided herein are methods and compositions for expression of a nucleic acid construct comprising nucleic acids encoding a) a recombinant polypeptide, and b) a prototrophy-restoring enzyme in a host cell that is auxotrophic for at least one metabolite. In various embodiments, the host cell is auxotrophic for a nitrogenous base compound or an amino acid. The invention involves introducing an analog into the growth media for the host cell such that the analog is incorporated into the recombinant polypeptide or a nucleic acid coding sequence thereof. In various embodiments, the compositions and methods disclosed herein result in improved recombinant protein expression compared to expression of recombinant protein in an antibiotic selection system, or compared to expression of the recombinant protein in an expression system that lacks a metabolite analog.

Production of recombinant proteins utilizing non-antibiotic selection methods and the incorporation of non-natural amino acids therein

The present invention provides an array for rapidly identifying a host cell population capable of producing a heterologous protein with improved yield and/or quality. The array comprises one or more host cell populations that have been genetically modified to increase the expression of one or more target genes involved in protein production, decrease the expression of one or more target genes involved in protein degradation, or both. One or more of the strains in the array may express the heterologous protein of interest in a periplasm compartment or may secrete the heterologous protein extracellularly through an outer cell wall. The strain arrays are useful for screening for improved expression of any protein of interest including therapeutic proteins, hormones, growth factors, extracellular receptors or ligands, proteases, kinases, blood proteins, chemokines, cytokines, antibodies and the like.

The invention is a process for improved production of a recombinant mammalian protein by expression in a Pseudomonad, particularly in aorganism. The process improves production of mammalian proteins, particularly human or human-derived proteins, over known expression systems such asin comparable circumstances Processes for improved production of isolated mammalian, particularly human, proteins are provided.

Provided herein are methods and compositions for expression of a nucleic acid construct comprising nucleic acids encoding a) a recombinant polypeptide, and b) a prototrophy-restoring enzyme in a host cell that is auxotrophic for at least one metabolite. In various embodiments, the host cell is auxotrophic for a nitrogenous base compound or an amino acid. The invention involves introducing an analogue into the growth media for the host cell such that the analogue is incorporated into the recombinant polypeptide or a nucleic acid coding sequence thereof. In various embodiments, the compositions and methods disclosed herein result in improved recombinant protein expression compared to expression of recombinant protein in an antibiotic selection system, or compared to expression of the recombinant protein in an expression system that lacks a metabolite analogue.

This invention is a process for improving the production levels of recombinant proteins or peptides or improving the level of active recombinant proteins or peptides expressed in host cells. The invention is a process of comparing two genetic profiles of a cell that expresses a recombinant protein and modifying the cell to change the expression of a gene product that is upregulated in response to the recombinant protein expression. The process can improve protein production or can improve protein quality, for example, by increasing solubility of a recombinant protein.

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Poway, CA, United States of America

Charles H Squires