Pfenex Inc.

Dow Global Technolgoies LLC

Dow Agrosciences LLC

Charles H. Squires

Diane M. Retallack

David C. Watkins

Frank H. Gaertner

Robert Shutter

Stacey Lynn Lee

Diane M Retallack

Charles H Squires

Russell J Coleman

Thomas Martin Ramseier

Jane C Schneider

Charles D Hershberger

Lawrence C Chew

David K Watkins

Frank H Gaertner

Hongfan Jin

Adam Curry Chapman

Torben Bruck

Kenneth Edwin Narva

Donald J Merlo

Aaron Todd Woosley

Thomas Meade

Sean Michael Russell

Sol M Resnick

Venkiteswaran Subramanian

Henry W Talbot

Torben R Bruck

Sol Resnick

Tom Meade

David C Watkins

Adam Chapman

Thomas M Ramseier

Russell John Coleman

Charles Douglas Hershberger

The present invention relates to the field of medicine, in particular, to the production of large amounts of a soluble recombinant polypeptide as part of a fusion protein comprising an N-terminal fusion partner linked to the polypeptide of interest.

Fusion partners for peptide production

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

strains that harbor transformation-enhancing genes on a plasmid capable of replication independently of thechromosome, the Ti plasmid, and plant transformation binary vectors, and uses for thesestrains are provided. Additionally,strains that are deficient in DNA recombination functions that result in instability or rearrangement of plant transformation binary vectors, and that harbor transformation-enhancing genes on a plasmid capable of replication independently of thechromosome, the Ti plasmid, and plant transformation binary vectors, and uses for these strains, are also provided. Further included arestrains that harbor transformation-enhancing genes integrated into thechromosome at a locus that does not interfere with or otherwise compromise the normal growth and plant transformation ability of thecells, and uses for thesestrains. Plants made using thesestrains are also described.

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.

The present invention relates to the field of recombinant toxin protein production in bacterial hosts. In particular, the present invention relates to production processes for obtaining high levels of a recombinant CRM197, Diphtheria Toxin, Pertussis Toxin, Tetanus Toxoid Fragment C, Cholera Toxin B, Cholera holotoxin, andExotoxin A, from a bacterial host.

High level expression of recombinant toxin proteins

Improved methods and prokaryotic expression systems for producing recombinant proteins using Sec-system secretion signal peptides are described usingSec secretion systems. Specific novel Sec-system secretion signal peptides are described, as are fusion proteins and coding sequences for improved secretion of recombinant proteins and peptides.

Expression systems with sec-system secretion

Compositions and methods for improving expression and/or secretion of a polypeptide of interest in a host cell are provided. Compositions including a coding sequence for a bacterial secretion signal peptide are provided. The compositions of the invention are useful for increasing accumulation of properly processed proteins in the periplasmic space of a host cell, or for increasing secretion of properly processed proteins. In particular, isolated secretion signal peptide-encoding nucleic acid molecules are provided. Additionally, amino acid sequences corresponding to the nucleic acid molecules are encompassed. The present invention provides for isolated nucleic acid molecules including nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24, and the nucleotide sequences set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23, as well as variants and fragments thereof.

Bacterial leader sequences for increased expression

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Poway, CA, United States of America

Diane M Retallack