Expression vectors for producing tag-cleavable fusion proteins in multiple expression systems

The mammalian protein expression system that Medicilon provides is based on a serum-free cultivated system to provide the gene expression optimization, transient expression, stable expression and antibody production and other eukaryotic protein services.

Researchers of Medicilon established a well-developed mammalian protein expression system and purification services platform, which offers the expression and purification services of a variety of recombinant protein , antibody or fragment of antibody. Email:marketing@medicilon.com.cn web:www.medicilon.com

A target polypeptide can be prepared by expressing a fusion protein including the target polypeptide and a protein tag that improves protein expression and facilitates protein purification. The protein tag can be removed by protease cleavage. A target polypeptide thus prepared, however, may be inactive due to possible addition of terminal amino acid residues.

Different expression systems might be needed for preparation of different target polypeptides. For example, glycopolypeptides are preferred to be expressed in mammalian cells, which possess glycosylation machinery. As another example, polypeptides toxic to mammalian cells can only be expressed in non mammalian expression systems. Therefore, a single expression system is not sufficient for producing a large variety of target polypeptides.

The present invention provides a kit containing multiple expression vectors for producing tag-cleavable fusion proteins in various expression systems, or a vector for producing fusion proteins in E. coli inclusion bodies. The multiple expression vectors can be categorized into two groups; namely, one for high-efficient cloning of a target gene, and the other for producing a sequence-specific target polypeptide (i.e., having the exact amino acid sequence encoded by a target gene).

In one aspect, this invention features a kit containing 2–6 expression vectors, at least two of which allow protein production in two different expression systems, such as an E. coli system, a yeast system (e.g., a pichica system), a baculovirus system, a mammalian cell system, or a cell-free system. Each of the expression vectors includes a first portion encoding a protein tag, and a second portion containing a nucleotide sequence that encodes a protease recognition site and includes one or two restriction sites identical in all of the 2–6 expression vectors. A fusion protein expressed therefrom include the protein tag, the protease recognition site, and the target polypeptide encoded by the target gene. Upon protease cleavage at the protease recognition site, the target polypeptide separates from the protein tag.

Any protein tag commonly used in expressing tag-fused proteins can be employed in the vectors described above. Exemplary tags include but are not limited to hexa-His, Maltose binding protein (MBP), N-utilizing substance A (NusA), thioredoxin, calmodulin-binding protein (CBP), glutathione S-transferase (GST), and α-factor.

A protease recognition site is a particular amino acid sequence recognizable by a protease, which cleaves within this sequence. Any commonly used protease recognition site can be employed in the vectors described above. Examples include but are not limited to the recognition sites of thrombin, Factor Xa (FXa), and tobacco etch virus (TEV) protease.

In one example, the kit of this invention contains 2–6 expression vectors, each of which includes the cloning sites of EcoR I and Xho I. These vectors can include a nucleotide sequence that encodes the recognition site of FXa. See FIG. 1, bottom panel. Such vectors include pHTPP10, pHTPP11 (or pHTPP11-EGFP), pHTPP12, pHTPP13, pHTPP14, pHTPP15, pHTPY2, pHTPBV5, pHTPM3, pHTPM6, and pHTPC1. They can also include a nucleotide sequence that encodes the recognition site of TEV protease. Such vectors include pHTPBV1, pHTPBV2, and pHTPBV4.

In another example, the kit of this invention contains 2–6 expression vectors, each of which includes the nucleotide sequence of GAGAACCTGTACGTACAG (encoding the TEV protease recognition site and including an SnaB I site for cloning a target gene). A fusion protein expressed from this vector can yield, upon TEV protease cleavage, a polypeptide having the exact amino acid sequence encoded by a target gene that contains its own stop codon. See FIG. 1, top panel. Examples of these vectors include pHTPP41, pHTPP42, pHTPP43, pHTPP44, pHTPP45, pHTPP46, pHTPY4, pHTPBV3, pHTPBV6, pHTPM5, and pHTPM7.

The kit as described above can further includes an expression vector for producing a fusion protein in inclusion bodies in E. coli. This fusion protein includes a target protein and a protein carrier, such as ketosteroid isomerase (KSI) and polyhedron, which facilitates translocation of the fusion protein to inclusion bodies in E. coli. Examples include pHTPI2, pHTPI2-EGFP, pHTPI3, and pHTPI3-EGFP.

All of the above-mentioned kits can be combined with each other to form additional new kits, which are also within the scope of this invention.

In another aspect, the present invention provides a kit containing a first vector set including two expression vectors as described above, both allowing production of tag-cleavage fusion proteins in a first expression system. Further, the first vector of this set includes one or two efficient cloning sites (e.g., EcoR I and Xho I) and the second vector includes the nucleotide sequence that encodes the TEV protease recognition site and includes an SnaB I site for cloning a target gene. These two expression vectors can include nucleotide sequences that encode a same protein tag. Exemplary sets of vectors contained in this kit include but are not limited to pHTPP10 and pHTPP41, pHTPP11 and pHTPP42, pHTPP12 and pHTPP43, pHTPP13 and pHTPP44, pHTPP14 and pHTPP45, pHTPP15 and pHTPP46, pHTPY2 and pHTPY4, and pHTPM3 or pHTPM6 and pHTPM5 or pHTPM7. As another example, one vector in the kit is pHTPBV1, pHTPBV2, pHTPBV4, or pHTPBV5; and the other is pHTPBV3 or pHTPBV6.

This kit can include a second set of two expression vectors as described above. This expression vector set allows protein expression in an expression system different from the first expression system. Similar to the first set, the second set includes one vector that has one or two efficient cloning sites (e.g., EcoR I and Xho I) and the other vector that contains the nucleotide sequence encoding the TEV protease recognition site and including an SnaB I site for cloning a target gene. The two expression vectors can include nucleotide sequences that encode the same protein tag.

Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appending claims.

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