Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-06-02
2002-12-31
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...
C435S183000, C435S254110, C435S254210, C435S320100, C435S069100, C435S069800, C536S023100, C536S023200, C536S023500, C536S023510, C536S023700, C536S023740, C530S333000
Reexamination Certificate
active
06500645
ABSTRACT:
FIELD OF INVENTION
The present invention relates to polypeptides produced in yeast, a DNA construct comprising a DNA sequence encoding such polypeptides, vectors carrying such DNA fragments and yeast cells transformed with the vectors, as well as a process of producing heterologous proteins in yeast.
BACKGROUND OF THE INVENTION
Yeast organisms produce a number of proteins synthesized intracellularly, but having a function outside the cell. Such extracelluar proteins are referred to as secreted proteins. These secreted proteins are expressed initially inside the cell in a precursor or a pre-form containing a pre-peptide sequence ensuring effective direction of the expressed product across the membrane of the endoplasmic reticulum (ER). The pre-peptide, normally named a signal peptide, is generally cleaved off from the desired product during translocation. Once entered in the secretory pathway, the protein is transported to the Golgi apparatus. From the Golgi the protein can follow different routes that lead to compartments such as the cell vacuole or the cell membrane, or it can be routed out of the cell to be secreted to the external medium (Pfeffer et al. (1987) Ann. Rev. Biochem. 56:829-852).
Several approaches have been suggested for the expression and secretion in yeast of proteins heterologous to yeast. European publication 088632A describes a process by which proteins heterologous to yeast are expressed, processed and secreted by transforming a yeast organism with an expression vector harbouring DNA encoding the desired protein and a signal peptide, preparing a culture of the transformed organism, growing the culture and recovering the protein from the culture medium. The signal peptide may be the desired protein's heterologous signal peptide, or a hybrid of a homologous and a heterologous signal peptide.
A problem encountered with the use of signal peptides heterologous to yeast may be that the heterologous signal peptide does not ensure efficient translocation and/or cleavage after the signal peptide.
The
Saccharomyces cerevisiae
MF&agr;1 (&agr;-factor) is synthesized as a pre-pro form of 165 amino acids comprising a 19 amino acids long signal- or pre-peptide followed by a 64 amino acids long “leader” or pro-peptide, (Kurjan et al. (1982) Cell 30:933-943). Use of signal/leader peptides homologous to yeast is described in U.S. Pat. No. 4,546,082; EP publications 0116201A, 0123294A, 0123544A, 0163529A, 0123289A, EP No. 0100561B, and PCT Publication WO 95/02059.
In EP 0123289A utilization of the
S. cerevisiae
&agr;-factor precursor is described whereas EP 0100561 describes the utilization of the
S. cerevisiae
PHO5 signal and WO 95/02059 describes the utilization of YAP3 signal peptide for secretion of foreign proteins.
U.S. Pat. No. 4,546,082 and European Publication Nos. 0016201A, 0123294A, 0123544A and 0163529A describe processes by which the &agr;-factor signal-leader from
S. cerevisiae
(MF&agr;1 or MF&agr;2) is utilized in the secretion process of expressed heterologous proteins in yeast. Secretion and processing of the desired protein was demonstrated by fusing a DNA sequence encoding the
S. cerevisiae
MF&agr;1 signal/leader peptide at the 5′ end of the gene for the desired protein.
EP 0206783 discloses a system for the secretion of polypeptides from
S. cerevisiae
whereby the &agr;-factor signal/leader sequence has been truncated to eliminate the four &agr;-factor peptides present on the native sequence so as to leave the signal/leader peptide itself fused to a heterologous polypeptide via the &agr;-factor processing site Lys-Arg-Glu-Ala-Glu-Ala (SEQ ID NO:93). It is indicated that this construction leads to an efficient process for production of smaller peptides (less than 50 amino acids). For the secretion and processing of larger polypeptides, the native &agr;-factor leader sequence has been truncated to leave one or two &agr;-factor peptides between the leader peptide and the polypeptide.
A number of secreted proteins are routed so that the precursor is exposed to a proteolytic processing system which can cleave the peptide bond at the carboxy end of two consecutive basic amino acids. This enzymatic activity is in
S. cerevisiae
encoded by the KEX 2 gene (Julius et al. (1984) Cell 37:1075). Processing of the product by the KEX 2 protease is needed for the secretion of active
S. cerevisiae
mating factor &agr;1 (MF&agr;1 or &agr;-factor) but is not involved in the secretion of active
S. cerevisiae
mating factor a.
Secretion and correct processing of a polypeptide intended to be secreted is obtained in some cases when culturing a yeast organism which is transformed with a vector constructed as indicated in the references given above. In many cases, however, the level of secretion is very low or there is no secretion, or the proteolytic processing may be incorrect or incomplete. As described in WO 90/10075, this is believed to be ascribable, to some extent, to an insufficient exposure of the processing site present between the C-terminal end of the leader peptide and the N-terminal end of the heterologous protein so as to render it inaccessible, or less accessible, to proteolytic cleavage, for example, by the KEX 2 protease.
WO 90/10075 describes a yeast expression system with improved processing of a heterologous polypeptide obtained by providing certain modifications near the processing site at the C-terminal end of the leader peptide and/or the N-terminal end of a heterologous polypeptide fused to the leader peptide.
SUMMARY OF THE INVENTION
The present invention describes modifications of the N-terminal end of the heterologous polypeptide designed as extensions which can be cleaved off either by naturally occurring yeast proteases before purification from the culture media or by in vitro proteolysis during or subsequently to purification of the product from the culture media.
In one aspect, the present invention is drawn to a DNA construct encoding a polypeptide having the structure:
signal peptide-leader peptide-X
1
-X
2
-X
3
-X
4
X
5
-X
6
-X
7
-heterologous protein
wherein
X
1
is Lys or Arg;
X
2
is Lys or Arg, X
1
and X
2
together defining a yeast processing site;
X
3
is Glu or Asp;
X
4
is a sequence of amino acids with the following structure
(A-B)
n
wherein A is Glu or Asp, B is Ala, Val, Leu or Pro, and n is 0 or an integer from 1 to 5, and when n≧2 each A and B is the same or different from the other A(s) and B(s); or
X
4
is a sequence of amino acids with the following structure
(C)
m
wherein C is Glu or Asp, and m is 0 or an integer from 1 to 5;
X
5
is a peptide bond or is one or more amino acids which may be the same or different;
X
6
is a peptide bond or an amino acid residue selected from the group consisting of Pro, Asp, Thr, Glu, Ala and Gly; and
X
7
is Lys or Arg.
A specific embodiment of the present invention is drawn to a DNA construct encoding a polypeptide having the structure:
signal peptide-leader peptide-X
1
-X
2
-X
3
-X
4
-X
5
-X
6
-X
7
-heterologous protein
wherein X
3
-X
4
-X
5
-X
6
-X
7
are the sequence Glu Glu Ala Glu Pro Lys (SEQ ID NO: 1).
The sequence Glu Glu Ala Glu Pro Lys (SEQ ID NO: 1) forms an extension at the N-terminal of the heterologous polypeptide. This extension not only increases the fermentation yield but is protected against dipeptidyl aminopeptidase (DPAP A) processing, resulting in a homogenous N-terminal of the polypeptide. The extension is constructed in such a way that it is resistant to proteolytic cleavage during fermentation so that the N-terminally extended heterologous protein product can be purified from the culture media for subsequent in vitro maturation, e.g. by trypsin or
Achromobacter lyticus
protease I. The desired in vitro removal of the N-terminal extension of SEQ ID NO:1 is readily achieved by either trypsin or
Achromobacter lyticus
protease I, presumably due to flexibility of the N-terminal extension peptide resulting in an improved yield of the matured heterologous protein.
Another specific embodiment of the present invention is drawn to a DNA construct
Balschmidt Per
Brandt Jakob
Havelund Svend
Kjeldsen Thomas Børglum
Pettersson Annette Frost
Bora Richard
Green, Esq. Reza
Hutson Richard
Novo Nordisk A S
Prouty Rebecca E.
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