Modified E. coli enterotoxin II signal peptide and a...

Details Modified E. coli enterotoxin II signal peptide and a... Modified E. coli enterotoxin II signal peptide and a...

2001-03-07

2003-08-12

Navarro, Mark (Department: 1645)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C530S326000, C530S350000

Reexamination Certificate

active

06605697

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a modified
E. coli
enterotoxin II signal peptide, a gene encoding said peptide, a vector comprising said gene fused with a gene encoding a heterologous protein, a microorganism transformed with said vector, and a process for producing the heterologous protein using said microorganism.
BACKGROUND OF THE INVENTION
Many heterologous proteins have been produced using genetically engineered host microorganisms by an intracellular method or secreting method.
In the intracellular method, a gene encoding a heterologous protein is expressed and accumulated in the cytoplasm of a microorganism. Although this method is known to give a relatively high heterologous protein yield, the expressed heterologous protein is not of a natural active form but methionylated at the N-terminus. Further, the biologically inactive heterologous protein produced by this method often forms insoluble inclusion bodies which must be solubilized and converted into a naturized, active form by a refolding process.
As to the secreting method, a gene encoding a fusion protein of a signal peptide and heterologous protein is expressed in the cytoplasm of a microorganism, and then, the fusion protein is processed by microorganism's signal peptidase to remove the signal peptide while passing through the cytoplasmic membrane. The processed protein is secreted into the periplasm space between the cytoplasmic (inner) membrane and outer membrane of the microorganism. However, this method is known to give a much lower yield of heterologous protein, as compared with the intracellular method. Therefore, there is a need for improving the productivity of the secreting method. In this line, it has been reported that accurate and efficient cleavage of the signal peptide moiety of an expressed fusion protein by signal peptidase is important in enhancing the yield of secreted heterologous protein (Akita, M. et al.,
J. Biol. Chem
., 265, 8164(1990)).
Generally, signal peptides are classified into two groups, hydrophilic signal peptides and hydrophobic signal peptides. A hydrophilic signal peptide is usually composed of 12 to 70 amino acids. A typical hydrophobic signal peptide, e.g.,
E. coli
enterotoxin II signal peptide, contains 13 to 30 amino acids, and it is comprised of three regions; an N-terminal hydrophilic region containing one or two basic amino acids; a central hydrophobic region containing about 10 basic amino acids; and a C-terminal hydrophilic region containing amino acids having small side-chains.
As a heterologous protein expressed in the form of a fusion protein with a signal peptide is often degraded rapidly by cytoplasmic proteinase, the yield of secreted heterologous protein decreases as the secretory efficiency of the signal peptide becomes low. Therefore, the yield of secreted heterologous proteins may be enhanced by modifying the signal peptide moiety of fusion proteins expressed in host microorganisms.
Human growth hormone (hGH) is composed of 191 amino acids and has a molecular weight of 21,500 Da. Since a purified form of hGH was first isolated from human pituitary in 1956 (Li and Papkoff,
Science
, 124, 1293(1956)), there have been made a large number of works on hGH to elucidate, e.g., the effect of hGH on human metabolism (Beck, J. C. et al.,
Science
, 125, 884(1957)) and inhibitory activity of hGH on pituitary nanocormia (Raben, M. S.,
J. Clin. Endocrinol
., 18, 901(1958)). Recently, it has been reported that hGH is also effective in the treatment of Turner's syndrome, osteoporosis, vulnus and burn.
As the amount of hGH obtained from human pituitary is limited, there has been an attempt to produce a large amount of hGH in genetically engineered
E. coli
by an intracellular method (Goeddel, D. V. et al.,
Nature
, 281, 544 (1979)). However, this method is hampered by the aforementioned problem of producing methionylated hGH which is not suitable for human application. A further attempt to remove methionine from the methionylated hGH using dipeptidyl aminopeptidase I resulted in an unacceptablly low yield of hGH.
Accordingly, the secretory production of natural hGH has been tried. For example, EP Nos 55942, 20147 and 114695 disclose methods for expressing a natural form of hGH and recovering it by secretion. However, the recoverable amount of hGH produced by these methods is only marginal.
EP No. 177,343 discloses a method for producing hGH, which comprises expressing a gene encoding a fusion protein of hGH and alkaline phosphatase or enterotoxin signal peptide, in the presence of an expression inducer, isopropylthio-&bgr;-D-galactoside (IPTG), and secreting hGH into periplasm. However, the method gives a low hGH yield and requires the use of the expensive expression inducer, IPTG.
Accordingly, there has been existed a need to develop a new efficient method for producing hGH in a high yield.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide a modified
E. coli
enterotoxin II signal peptide which can be advantageously used in a secreting method of producing a heterologous protein to enhance secretion efficiency.
Another object of the present invention is to provide a gene encoding said peptide.
A further object of the present invention is to provide a vector comprising said gene fused with a gene encoding heterologous protein.
A further object of the present invention is to provide a microorganism transformed with said vector.
A further object of the present invention is to provide a process for producing a hererologous protein using said microorganism.
In accordance with one aspect of the present invention, there is provided a modified
E. coli
enterotoxin II signal peptide (designated MST) characterized in that at least one of the 2nd, 4th, 5th, 12th, 20th and 22nd amino acids of
E. coli
enterotoxin II signal peptide represented by the following amino acid sequence (SEQ ID NO: 1) is replaced by another amino acid, with the proviso that at least one of the 2nd and 4th amino acid of the MST is lysine:
Met Lys Lys Asn Ile Ala Phe Leu Leu Ala Ser Met
                  5                  10

Phe Val Phe Ser Ile Ala Thr Asn Ala Tyr Ala
         15                  20


REFERENCES:
Rudinger et al (Peptide HormonesUniversity Park Press, p. 6), 1976.*
Burgess et al (J. of Cell Biology vol. III pp 2129-2138), Nov. 1990.*
Lazar et al (Molecular & Cellular Biology vol. 8(3) pp 1247-1252), Mar. 1988.*
Joblin et al (Molecular Microbiology vol. 5(7) pp 1755-1767), 1991.

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