Methods and compositions for enhanced protein expression and purification
Inventors
Butt, Tauseef R. • Panavas, Tadas • Karwa, Amolkumar • Peroutka, Raymond J. • Marblestone, Jeffrey G.
Assignees
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Publication Number
US-8691958-B2
Publication Date
2014-04-08
Expiration Date
Abstract
Methods for enhancing expression levels, secretion, and purification of heterologous fusion proteins in a host cell are disclosed.
Core Innovation
The patent describes engineered SUMO tags (SUMO* variants) with altered SUMO protease interaction domains that resist cleavage by wild-type SUMO proteases in eukaryotic cells. This altered interaction prevents cleavage of the SUMO* fusions in yeast, insect, and mammalian contexts, while enabling enhanced expression and solubility of heterologous fusion proteins. The document further indicates that these SUMO* fusions can support improved secretion and/or purification of the heterologous protein of interest.
The patent also describes engineered SUMO proteases that specifically cleave the engineered SUMO tags. These engineered SUMO proteases are defined by a SUMO interaction domain comprising an amino acid sequence pattern, including WLNX1X2X3X4X5 (SEQ ID NO:6), and engineered variants are specified by sequence pattern constraints and homology to reference sequences (SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5). In this system, cleavage by the engineered protease releases the protein of interest and enables generation of desired N-termini.
Beyond the fusion and protease components, the document describes nucleic acids encoding engineered SUMO and engineered SUMO proteases, together with expression vectors, isolated cells containing the expression vector, and optional affinity tags for purification. The document further describes use of engineered SUMO components in providing peptide/protein libraries and microarrays, and describes kits and related compositions comprising engineered SUMO, optional affinity tags, and engineered SUMO proteases.
Claims Coverage
The partial content provides two independent claims. Each independent claim focuses on nucleic acids encoding an engineered SUMO protease, with defining sequence features in the SUMO interaction domain and specific homology requirements or fixed residue configurations, respectively.
Engineered SUMO protease SUMO interaction domain pattern with variable residues and homology threshold
An isolated nucleic acid molecule encoding an engineered SUMO protease having a SUMO interaction domain comprising amino acid sequence WLNX1X2X3X4X5 (SEQ ID NO:6), wherein X1 and X5 are any non-acidic amino acid and X2, X3, and X4 are any amino acid, and wherein the engineered SUMO protease has at least 80% homology with an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
Engineered SUMO protease SUMO interaction domain with specific residue configuration
An isolated nucleic acid molecule encoding an engineered SUMO protease having a SUMO interaction domain comprising amino acid sequence WLNX1X2X3X4X5 (SEQ ID NO:6) where X1–X5 are specified as: serine, glycine, isoleucine, isoleucine, and serine; alanine, methionine, isoleucine, isoleucine, and alanine; serine, threonine, isoleucine, isoleucine, and alanine; or serine, threonine, isoleucine, isoleucine, and methionine.
Across the independent claims, coverage centers on nucleic acids encoding engineered SUMO proteases defined by a SUMO interaction domain sequence pattern (SEQ ID NO:6). One claim requires at least 80% homology to one of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 while allowing variable residue selection at X1–X5 under the non-acidic constraint at X1 and X5. The other narrows the SEQ ID NO:6 variant space by fixing X1–X5 to specific residue configurations.
Stated Advantages
Enhanced expression and solubility of heterologous fusion proteins via engineered SUMO* fusions.
Improved secretion and/or purification of heterologous fusion proteins using engineered SUMO*.
Specific cleavage of engineered SUMO tags by engineered SUMO proteases to release the protein of interest.
Generation of desired N-termini of the protein of interest after cleavage.
Documented Applications
Expression in bacterial and eukaryotic hosts using engineered SUMO and engineered SUMO proteases.
Purification and cleavage in vitro or in vivo using engineered SUMO* and engineered SUMO proteases.
Use in peptide/protein libraries and microarrays.
Kit or vector-based workflows comprising engineered SUMO, optional affinity tags, and engineered SUMO proteases.
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