US 9,856,483 B2Granted

Expression system for producing protein having a N-terminal pyroglutamate residue

OwnerAndrew H. -J. Wang, Kai-Fa Huang +1inventors

InventorsAndrew H. -J. Wang, Kai-Fa Huang, Yan-Ping Shih

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Abstract

Disclosed herein is an expression system and uses thereof. The expression system comprises two vectors that express two fusion proteins and a glutaminyl cyclase (QC) with E45Q mutation, so as to autonomously produce a target protein having a N-terminal pyroglutamate (pGlu) residue in a host cell. The first fusion protein is composed of a maltose binding protein (MBP) and a tobacco etch virus protease (TEVP); and the second fusion protein includes in sequence, a thioredoxin, a S-tag, a linker having a TEVP recognition site therein, a target protein, and a (His)-6-tag. The second fusion protein is cleaved by the TEVP that is expressed by the first fusion protein, and then catalyzed by QC with E45Q mutation so as to autonomously produce the target protein having a N-terminal pGlu residue in the host cell.

Claims20

Dependent claims

An expression system for producing a target protein having a N-terminal pyroglutamate (pGlu) residue in a host cell, comprising:

(a) a first vector comprising a first nucleotide sequence encoding a first fusion protein, from the N-terminus to C-terminus, a maltose binding protein (MBP) and a tobacco etch virus protease (TEVP);
and
(b) a second vector comprising in sequence,
(b-1) a second nucleotide sequence encoding a second fusion protein, from the N-terminus to C-terminus, a thioredoxin (Trx), a S-tag, a linker having a TEVP recognition site (rsTEV) therein, the target protein, and a (His)-6-tag;
and
(b-2) a third nucleotide sequence encoding a glutaminyl cyclase (QC) having a E45Q mutation;
wherein,
the second nucleotide sequence is characterized in having two restriction endonuclease cleavage sites respectively located within the linker, and between the target protein and the (His)6-tag;
and
the N-terminal pGlu residue of the target protein is autonomously formed in the host cell.

7 more elements

Dependencies are inferred from the claim text.

Facts

Application: 14/661,052Filed 18 Mar 2015
Published: US 2016/0273015 A122 Sep 2016
Granted: 2 Jan 2018
Term ends: Not estimatedThe source records don’t support an estimate.

Classification12 codes

C12N 15/70
Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression: vectors or expression systems…
C07K 14/52
From animals; from humans: cytokines; Lymphokines; Interferons
C12N 9/104
Acyltransferases: aminoacyltransferases
C12P 21/02
Preparation of peptides or proteins: having a known sequence of two or more amino acids, e.g. glutathione