Rapidly cleavable sumo fusion protein expression system

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A recombinant expression system for the expression of a poly amino acid, peptide or protein is provided. The poly amino acid of interest is expressed as a fusion protein that includes an amino acid sequence recognized and cleaved by a Ulp1 protease. The amino acid sequence joined to the poly amino acid of interest is preferably from a SUMO (small ubiquitin-like molecule) protein. This sequence imparts favorable solubility and refolding properties to the fusion protein. A purification tag may also be incorporated into the fusion protein for ease of isolation. The Ulp1 protease used to cleave the fusion protein may be the Ulp1 protease or the active Ulp1 protease fragment, Ulp1(403-621). The Ulp1 protease rapidly and specifically cleaves the fusion proteins of the invention at the Ulp1 cleavage site. The amino acid sequence recognized by a Ulp1 protease is cleaved asymetrically to leave only an N-terminal serine joined to the poly amino acid of interest. This recombinant expression system is particularly advantageous for expression and rapid and highly specific cleavage and purification of poly amino acids that have low solubilities or are difficult to express in other systems.

Numerous recombinant expression systems are available for production of foreign proteins. See for example Ausubel et al. (Eds.), Current Protocols in Molecular Biology, Wiley, N.Y. (1999); Wu, R. (Ed.), Recombinant DNA methodology II, Academic Press, NY (1995). One problem common to the available expression systems is that it is difficult to efficiently express many foreign proteins in active form at high levels. Another difficulty arises when the expression of the protein of interest leads to precipitation of the protein as an insoluble amorphous mass in the host cell bearing the expression vector. There remains a need for an efficient expression system, especially for proteins that are difficult to express. Optimally, the expression system should provide high levels of soluble, correctly folded, or active recombinant peptides or proteins that may be easily purified from the expression system.

The present invention provides a recombinant fusion protein that comprises an expression enhancing amino acid sequence cleavable by Ulp1 protease or an active fragment of Ulp1 protease (for example the fragment from amino acid positions 403 to 621) and a recombinant poly-amino acid of interest, particularly one that is difficult to express in a recombinant expression system. The fusion protein may also include a purification tag for ease of isolation.

The invention further provides a method of expressing a recombinant poly-amino acid of interest that is difficult to express in a recombinant expression system, by the steps of providing a vector encoding a fusion protein that includes the recombinant poly-amino acid of interest, preferably located C-terminally to a purification tag. The purification tag and the recombinant poly-amino acid of interest are separated by an expression enhancing amino acid sequence cleavable by Ulp1 protease or an active fragment of Ulp1 protease, such as the fragment from the amino acid residue at position 403 to the amino acid residue at position 621. The fusion protein is expressed from the vector in a suitable recombinant host cell.

The invention also provides a method for purifying a poly-amino acid of interest by providing a vector encoding a recombinant fusion protein that comprises an expression enhancing amino acid sequence cleavable by Ulp1 protease or an active fragment of the Ulp1 protease (for example the fragment from amino acid position 403 to 621) and the recombinant poly-amino acid of interest; expressing the fusion protein from the vector in a suitable recombinant host cell; and purifying the fusion protein by means of the purification tag and cleaving the purified fusion protein with a Ulp1 protease or Ulp1 protease fragment.

Also provided are expression vectors encoding the recombinant fusion proteins that comprise an expression enhancing amino acid sequence cleavable by Ulp1 protease or an active fragment of Ulp1 protease (for example the fragment from amino acid position 403 to 621) and a recombinant poly-amino acid of interest expressed from an efficient promoter.

In another embodiment of the present invention, host cells carrying the expression vectors encoding recombinant fusion proteins that include an expression enhancing amino acid sequence cleavable by Ulp1 protease or an active fragment of Ulp1 protease (for example the fragment from amino acid position 403 to 621) and a recombinant poly-amino acid of interest are provided.

Also provided are kits and products comprising: a recombinant vector encoding a fusion protein comprising a purification tag, an expression enhancing amino acid sequence cleavable by Ulp1 protease or an active fragment of Ulp1 protease (for example the fragment from amino acid position 403 to 621) and a multiple cloning site suitable for cloning a nucleic acid sequence encoding a poly-amino acid of interest, wherein the poly-amino acid of interest is difficult to express in a recombinant system; and further comprising a Ulp1 protease preparation. The kit may further comprise an antibody specific for the expression enhancing amino acid sequence cleavable by Ulp1 protease or an active fragment of Ulp1 protease (for example the fragment from amino acid position 403 to 621).

In yet another embodiment, the present invention provides an active fragment of the Ulp1 protease. Preferably, the active fragment of the Ulp1 protease comprises amino acid residues 403-621 of Ulp1. More preferably still, the active fragment of the Ulp1 protease consists essentially of amino acid residues 403-621 of Ulp1.

The present invention yet further provides nucleic acid molecules, including DNA and RNA molecules that include a nucleotide sequence encoding an expression enhancing amino acid sequence cleavable by Ulp1 protease or an active fragment of Ulp1 protease. The nucleic acid sequence encoding the expression enhancing amino acid sequence cleavable by Ulp1 protease or an active fragment of Ulp1 protease may also encompasses a restriction enzyme recognition site downstream of the nucleotide sequence encoding the expression enhancing amino acid sequence cleavable by Ulp1 protease or Ulp1 protease fragment. The restriction enzyme recognition site included in the nucleic acid molecule being suitable for cloning a nucleotide sequence encoding a poly-amino acid of interest. The nucleic acid molecules of the invention are particularly useful for the expression and purification of poly-amino acid of interest that are difficult to express in recombinant expression systems.