Compositions and methods for protein secretion

Details Compositions and methods for protein secretion Compositions and methods for protein secretion

1998-05-28

2002-01-01

Cochrane Carlson, Karen (Department: 1653)

Chemistry: molecular biology and microbiology

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

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

C435S069700, C435S252300, C435S320100, C530S350000, C530S300000, C536S023100, C536S023400, C536S023700

Reexamination Certificate

active

06335178

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to compositions and methods for secretion of functional proteins in a soluble form by host cells. In particular, the invention relates to proteins involved in targeting expression of a protein of interest extracellularly and to the periplasm, thus facilitating generation of a functional soluble protein.
BACKGROUND OF THE INVENTION
Proteins having clinical or industrial value may be obtained using techniques which facilitate their synthesis in bacterial or in eukaryotic cell cultures. However, once synthesized, there are often problems in recovering these recombinant proteins in substantial yields and in a useful form. For example, recombinant proteins expressed in bacteria often accumulate in the bacterial cytoplasm as insoluble aggregates known as inclusion bodies [Marston, (1986) Biochem. J. 240:1-12; Schein (1989) Biotechnology 7:1141-1149]. Similarly, recombinant transmembrane proteins which contain both hydrophobic and hydrophilic regions are intractable to solubilization.
While transmembrane recombinant proteins and recombinant proteins which are expressed in the cytoplasm may be solubilized by use of strong denaturing solutions (e.g., urea, guanidium salts, detergents, Triton, SDS detergents, etc.), solubilization efficiency is nevertheless variable and there is no general method of solubilization which works for most proteins. Additionally, many proteins which are present at high concentrations precipitate out of solution when the solubilizing agent is removed. Yet a further drawback to solubilization of recombinant proteins is that denaturing chemicals (e.g., guanidium salts and urea) contain reactive primary amines which swamp those of the protein, thus interfering with the protein's reactive amine groups.
Thus, what is needed is a method for producing soluble proteins.
SUMMARY OF THE INVENTION
The present invention provides a recombinant polypeptide comprising at least a portion of an amino acid sequence selected from the group consisting of SEQ ID NOs:47 and 49, SEQ ID NO:7 and variants and homologs thereof, and SEQ ID NO:8 and variants and homologs thereof.
This invention further provides an isolated nucleic acid sequence encoding at least a portion of an amino acid sequence selected from the group consisting of SEQ ID NOs:47 and 49, SEQ ID NO:7 and variants and homologs thereof, and SEQ ID NO:8 and variants and homologs thereof. In one preferred embodiment, the nucleic acid sequence is contained on a recombinant expression vector. In a more preferred embodiment, the expression vector is contained within a host cell.
Also provided by the present invention is a nucleic acid sequence that hybridizes under stringent conditions to a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:7 and variants and homologs thereof, and SEQ ID NO:8 and variants and homologs thereof.
The invention additionally provides a method for expressing a nucleotide sequence of interest in a host cell to produce a soluble polypeptide sequence, the nucleotide sequence of interest when expressed in the absence of an operably linked nucleic acid sequence encoding a twin-arginine signal amino acid sequence produces an insoluble polypeptide, comprising: a) providing: i) the nucleotide sequence of interest encoding the insoluble polypeptide; ii) the nucleic acid sequence encoding the twin-arginine signal amino acid sequence; and iii) the host cell, wherein the host cell comprises at least a portion of an amino acid sequence selected from the group consisting of SEQ ID NOs:47 and 49, SEQ ID NO:7 and variants and homologs thereof, and SEQ ID NO:8 and variants and homologs thereof, b) operably linking the nucleotide sequence of interest to the nucleic acid sequence to produce a linked polynucleotide sequence; and c) introducing the linked polynucleotide sequence into the host cell under conditions such that the fused polynucleotide sequence is expressed and the soluble polypeptide is produced.
Without intending to limit the location of the insoluble polypeptide, in one preferred embodiment, the insoluble polypeptide is comprised in an inclusion body. In another preferred embodiment, the insoluble polypeptide comprises a cofactor. In a more preferred embodiment, the cofactor is selected from the group consisting of iron-sulfur clusters, molybdopterin, polynuclear copper, tryptophan tryptophylquinone, and flavin adenine dinucleotide.
Without limiting the location of the soluble polypetide to any particular location, in one preferred embodiment, the soluble polypeptide is comprised in periplasm of the host cell. In an alternative preferred embodiment, the host cell is cultured in medium, and the soluble polypeptide is contained in the medium.
The methods of the invention are not intended to be limited to any particular cell. However, in one preferred embodiment, the cell is
Escherichia coli
. In a more preferred embodiment, the
Escherichia coli
cell is D-43.
It is not intended that the invention be limited to a particular twin-arginine signal amino acid sequence. In a preferred embodiment, the twin-arginine signal amino acid sequence is selected from the group consisting of SEQ ID NO:41 and SEQ ID NO:42.
The invention further provides a method for expressing a nucleotide sequence of interest encoding an amino acid sequence of interest in a host cell, comprising: a) providing: i) the host cell; ii) the nucleotide sequence of interest; iii) a first nucleic acid sequence encoding twin-arginine signal amino acid sequence; and iv) a second nucleic acid sequence encoding at least a portion of an amino acid sequence selected from the group consisting of SEQ ID NOs:47 and 49, SEQ ID NO:7 and variants and homologs thereof, and SEQ ID NO:8 and variants and homologs thereof; b) operably fusing the nucleotide sequence of interest to the first nucleic acid sequence to produce a fused polynucleotide sequence; and c) introducing the fused polynucleotide sequence and the second nucleic acid sequence into the host cell under conditions such that the at least portion of the amino acid sequence selected from the group consisting of SEQ ID NOs:47 and 49, SEQ ID NO:7 and variants and homologs thereof, and SEQ ID NO:8 and variants and homologs thereof is expressed, and the fused polynucleotide sequence is expressed to produce a fused polypeptide sequence comprising the twin-arginine signal amino acid sequence and the amino acid sequence of interest.
The location of the expressed amino acid sequence of interest is not intended to be limited to any particular location. However, in one preferred embodiment, the expressed amino acid sequence of interest is contained in periplasm of the host cell. In a particularly preferred embodiment, the expressed amino acid sequence of interest is soluble. Also without intending to limit the location of the expressed amino acid sequence of interest, in an alternative preferred embodiment, the host cell is cultured in medium, and the expressed amino acid sequence of interest is contained in the medium. In a particularly preferred embodiment, the expressed amino acid sequence of interest is soluble.


REFERENCES:
patent: 4683195 (1987-07-01), Mullis
patent: 4683202 (1987-07-01), Mullis et al.
Database GenBank on STN, Accession No. H65188. “yigU protein—Escherichia coli(strain K12)” Blattner et al. (1997).*
Database GenBank on STN, Accession No. P27857. “YigU_E. coli” Daniels et al. (1997).*
Database GenBank on STN, Accession No. A65189. “yigW protein—Escherichia coli” Blattner et al. (1997).*
Database GenBank on STN, Accession No. P27859. “YigW_E. coli” Daniels et al. (1997).*
Database GenBank on STn, Accession No. G65188. “hypothetical 15.6 kD protein in udp-rfaH intergenic region—Escherichia coli(strain K-12)” Blattner et al. (1997).*
Database GenBank on STN, Accession No. E65188. “hypothetical 11.3 kD protein in udp-rfaH intergenic region—Escherichia coli(strain K-12)” Blattner et al. (1997).*
Weiner et al. (Apr. 13, 1998) A Novel Ubiquitous System for Membrane Targeting a

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