Production of secreted polypeptides

Details Production of secreted polypeptides Production of secreted polypeptides

2001-11-14

2003-11-04

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S252300, C435S252330, C435S183000, C435S071100

Reexamination Certificate

active

06642027

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the increased production of proteins, preferably, heterologous secreted proteins and cells having interrupted peptide transport activity.
BACKGROUND
Secretion of heterologous polypeptides is a widely used technique in industry. A cell can be transformed with a nucleic acid encoding a heterologous polypeptide of interest to be secreted and thereby produce large quantities of desired polypeptides. This technique can be used to produce a vast amount of polypeptide over what would be produced naturally. Polypeptides of interest have a number of industrial applications, including therapeutic and agricultural uses, as well as use in foods, cosmetics, cleaning compositions, animal feed, etc.
Thus, increasing secretion of polypeptides is of interest. Secretion of polypeptides into periplasmic space or into their culture media is subject to a variety of parameters. Typically, vectors for secretion of a polypeptide of interest are engineered to position DNA encoding a secretory signal sequence 5′ to the DNA encoding the DNA of interest.
Attempts to increase secretion have often fallen into one of the following three areas: trying several different signal sequences, mutating the signal sequence, and altering the secretory pathway within the host. While some success has been found with the above methods, generally, they are time consuming and novel methods are desirable. Therefore, a problem to be solved is how to produce and/or secrete more proteins without solely relying on altering the signal sequence.
The instant invention provides a novel approach to improving secretion of polypeptides in a cell. Also provided herein are novel compositions useful in the methods of polypeptide secretion provided herein, and methods of making such compositions.
SUMMARY OF THE INVENTION
In one aspect of the invention, a method of increasing secretion of a polypeptide in a cell is provided. In a preferred embodiment, said cell selected would express at least one peptide transport protein. In one embodiment, the method comprises inactivating said at least one peptide transport protein in said cell and culturing said cell under conditions suitable for expression and secretion of said polypeptide.
The methods provided herein are applicable for production or secretion of polypeptides in a variety of cell types. For example, the cell can be selected from the group consisting of a plant cell, a fungal cell, a gram-negative microorganism and a gram-positive microorganism. In one embodiment, said cell is a gram-negative microorganism, preferably, a member of the family Escherichia. In another embodiment, said cell is a gram-positive microorganism, preferably a member of the family Bacillus. In a preferred embodiment, said cell is a gram-positive microorganism and is a member of the family Bacillus wherein said member of the family Bacillus is selected from the group consisting of
B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. thuringiensis, B. methanolicus
and
B. anthracis.
The polypeptide which is secreted or produced by the methods provided herein can be any polypeptide. In a preferred embodiment, it is a heterologous polypeptide. In one aspect, the polypeptide is selected from the group consisting of hormone, enzyme, growth factor and cytokine. In one embodiment, the polypeptide is an enzyme, preferably selected from the group consisting of proteases, carbohydrases, reductases, lipases, isomerases, transferases, kinases, phophatases, cellulase, endo-glucosidase H, oxidase, alpha-amylase, glucoamylase, lignocellulose hemicellulase, pectinase and ligninase. In one aspect, said polypeptide is a bacillus protease, preferably subtilisin. In another aspect, said polypeptide is an amylase, preferably, bacillus amylase.
The peptide transport protein can be a variety of proteins and can be inactivated in a variety of ways. In one aspect, the peptide transport protein is a gene product of a dciA operon, and preferably, is the gene product of the dciAE gene. The protein can be inactivated at the protein or nucleic acid level. In one aspect, the protein is inactivated because the gene encoding said protein has been mutated. In another embodiment, the operon comprising said gene has been mutated. The mutation can be caused in a variety of ways including one or more frameshifts, insertions, substitutions and deletions, or combinations thereof. The deletion can be of a single nucleotide or more, including deletion of the entire gene.
In another aspect of the invention, a method for producing a polypeptide in a cell is provided which comprises the steps of obtaining a cell comprising nucleic acid encoding a polypeptide to be produced, said cell further comprising a peptide transport operon wherein at least one gene product of said operon is inactive in said cell, and culturing said cell under conditions suitable for expression such that said polypeptide is produced. Preferably, the peptide transport operon is a dciA operon. The gene product of said operon can be inactivated at the nucleic acid or protein level. Preferably, the inactivated gene product is encoded by dciAA or dciAE.
In another aspect of the invention provided herein is a cell comprising a peptide transport operon, wherein said operon has been mutated such that said cell has increased polypeptide secretion. In a preferred embodiment, said operon is a dciA operon. In one embodiment, said operon has been mutated to inactivate a gene product of a dciAA and/or dciAE gene.


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