Fusion polypeptides comprising one or more inclusion body tags, methods and uses
Inventors
JONG, Wouter Simon Petrus • Luirink, Joen
Assignees
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Publication Number
US-11124555-B2
Publication Date
2021-09-21
Expiration Date
Abstract
A fusion polypeptide, comprising an inclusion body tag fused to at least one polypeptide of interest, is provided. The inclusion body tag comprises an inclusion body forming (IBF) amino acid sequence selected from X14X15AX17X18X19GLTVA GMLG and sequences having at least 71% identity thereto. Also provided are inclusion bodies comprising the fusion polypeptide, as well as nucleic acids, expression vectors, host cells and methods for its production.
Core Innovation
The disclosure relates to fusion polypeptides in which an inclusion body forming (IBF) tag is fused to at least one polypeptide of interest. The IBF tag is based on an inclusion body forming amino acid sequence derived from the E. coli TorA signal sequence (ssTorA/TorA), including core motif sequences such as SEQ ID NO:1 (GLTVAGMLG) and variant motifs defined by specified amino-acid patterns and identity thresholds.
The described IBF tag sequences include amino-acid sequence variants with at least 71% identity to the motif defined in the claim, as well as tag embodiments that comprise SEQ ID NO:3 or SEQ ID NO:44 with defined truncations/deletions corresponding to positions 29-36. The disclosure also supports fusion constructs that incorporate two or more IBF tags, with the tag being positioned C-terminal to the polypeptide of interest.
The disclosure further provides nucleic acids, expression vectors, and microbial host cells for expressing the fusion protein to form inclusion bodies, and it supports expressing fusion polypeptides and forming inclusion bodies. The document demonstrates inclusion body formation for multiple proteins, including toxic, unstable, and large proteins, and reports performance comparisons versus a KSI tag, including support for universal IB induction even for soluble proteins and for protection from proteolysis and toxicity.
Claims Coverage
Independent claim clm-00001 covers fusion polypeptides with an inclusion body tag comprising an IBF amino-acid sequence selected from specified sequence motifs and identity/variant constraints, fused to at least one polypeptide of interest. The claim includes three alternative structural options regarding the number of tags and the tag position or defined truncations of particular tag sequences.
Inclusion body tag with defined IBF motif selection and identity/variant constraints
A fusion polypeptide comprising an inclusion body tag fused to at least one polypeptide of interest, wherein said inclusion body tag comprises an inclusion body forming (IBF) amino acid sequence selected from: (i) X14X15AX17X18X19GLTVAGMLG (SEQ ID NO:1) with X14 selected from F and Y, X15 selected from L and M, X17 selected from Q and E, X18 selected from L, V and I, and X19 selected from G and V; and (ii) an amino acid sequence which has at least 71% identity to the sequence defined in i).
Two or more inclusion body tags or C-terminal tag placement or defined ssTorA truncation/deletion
Wherein: (a) the fusion polypeptide comprises two or more of the inclusion body tags; or (b) the inclusion body tag is C-terminal to said polypeptide of interest; or (c) the inclusion body tag comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:44 lacking one or more of the amino acids corresponding to positions 29-36 of SEQ ID NO:3 or SEQ ID NO:44.
Overall, the claim coverage centers on fusing a TorA-derived IBF motif tag or a variant with at least 71% identity to the defined motif to a polypeptide of interest, with further scope defined by using two or more tags, placing the tag C-terminally, or using SEQ ID NO:3 or SEQ ID NO:44 with deletions or truncations limited to positions 29-36.
Stated Advantages
Universal IB induction even for soluble proteins.
Protection from proteolysis and toxicity.
Simplified recovery.
Compatibility with N- or C-terminal placement and multiple tag copies.
Documented Applications
Inclusion body formation demonstrated for proteins including toxic, unstable, and large proteins, including comparison to a KSI tag.
Use of the fusion polypeptides to form inclusion bodies as described for a range of example proteins, including hEGF, IL-3, TrxA, MBP, CAT, TorA(HA), SymE, Hbp(Δss), GFP, and KSI.
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