Rapidly cleavable sumo fusion protein expression system for...

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

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C435S320100, C424S094630, C536S023400

Reexamination Certificate

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06872551

ABSTRACT:
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.

REFERENCES:
patent: 5643758 (1997-07-01), Guan et al.
Yoshinobu Ichimura, Takayoshi Kirisako, Toshifumi Takao, Yoshinori Satomi, Yasutsugu Shimonishi, Naotada Ishihara, Noboru Mizushima, Isei Tanida, Eiki Kominami, Mariko Ohsumi, Takeshi Noda and Yoshinori Ohsumi, “A ubiquitin-like system mediates protein lipidation”,Nature(2000) 408: 488-492.
Elena Mossessova and Christopher D. Lima, “Ulp1-SUMO Crystal Structure and Genetic Analysis Reveal Conserved Interactions and a Regulatory Element Essential for Cell Growth in Yeast”,Molecular Cell(2000) 5: 865-876.
Edward T.H. Yeh, Limin Gong, Tetsu Kamitani, “Ubiquitin-like proteins: new wines in new bottles”,Gene(2000) 248: 1-14.
Shyr-Jiann Li and Mark Hochstrasser, “The YeastULP2(SMT4) Gene Encodes a Novel Protease Specific for the Ubiquitin-Like Smt3 Protein”,Molecular and Cellular Biology(2000) 20: 2367-2377.
Katsunori Tanaka, Junko Nishide, Koei Okazaki, Hiroaki Kato, Osami Niwa, Tsuyoshi Nakagawa, Hideyuki Matsuda, Makoto Kawamukai, and Yota Murakami, “Characterization of a Fission Yeast SUMO-1 Homologue, Pmt3p, Required for Multiple Nuclear Events, Including the Control of Telomere Length and Chromosome Segregation”,Molecular and Cellular Biology(1999) 19: 8660-8672.
Shyr-Jiann Li and Mark Hochstrasser, “A new protease required for cell-cycle progression in yeast”,Nature(1999) 398: 246-251.
David P. Lane and Peter A. Hall, “SUMO-1: wrestling with a new ubiquitin-related modifier”,TIBS(1997) 22:374-376.
Erica S. Johnson, Ingrid Schwienhorst, R. Jürgen Dohmen, and Günter Blobel, “The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aoslp/Uba2p heterodimer”,The EMBO Journal(1997) 16: 5509-5519.

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