Intein-mediated protein splicing
OwnerNarendra S. Yadav, Jianjun Yanginventors
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Abstract
The present invention provides methods for intein-mediated protein splicing, particularly in plants. This permits in vivo and in vitro synthesis of homogeneous and large multi-functional hybrid protein polymers and circular proteins. Additionally, methods are provided which are suitable for the regulation of transgene expression, such that a particular transgene is expressed only under selected environmental conditions, in selected plant tissues, at selected development stages, or in selected plant generations.
Claims57
An isolated polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising an ExtN, a ExtC, and an Int interposed between said ExtN and said ExtC, wherein:
- said ExtN is the N-terminal portion of the polypeptide;
- said Int is an intein;
- and
- said ExtC is the C-terminal portion of the polypeptide,
- and wherein at least a portion of said nucleotide sequence has been modified to contain plant optimized codons.
An isolated polynucleotide comprising a nucleotide sequence that encodes a fusion polypeptide consisting of an ExtN, a ExtC, and an Int interposed between said ExtN and said ExtC, wherein:
- said ExtN is the N-terminal portion of the polypeptide;
- said Int is an intein;
- and
- said ExtC is the C-terminal portion of the polypeptide.
An isolated polynucleotide comprising a nucleotide sequence that encodes a polypeptide selected from the group consisting of:
- an ExtN and an IntN;
- and
- an ExtC and an IntC,
- wherein said IntN and said IntC together form a naturally split intein.
A method for producing a protein comprising an ExtN and a ExtC, said method comprising:
- (a) obtaining an N-nucleotide sequence that encodes an N-polypeptide comprising an ExtN and an IntN;
- (b) obtaining a C-nucleotide sequence that encodes a C-polypeptide comprising an IntC and an ExtC;
- (c) transforming a plant host with said N-nucleotide sequence and said C-nucleotide sequence such that said plant produces said protein;
- and
- (d) optionally recovering said protein.
A method for producing a protein that comprises an ExtN and a ExtC, said method comprising:
- (a) transforming an N-plant host with an N-polynucleotide comprising an N-nucleotide sequence that encodes an N-polypeptide comprising said ExtN and an IntN, such that said N-plant host produces said N-polypeptide;
- (b) transforming a C-plant host with a C-polynucleotide comprising a C-nucleotide sequence that encodes a C-polypeptide comprising a IntC and said ExtC, such that said C-plant host produces said C-polypeptide;
- and
- (c) crossing said N-plant host and said C-plant host to obtain a progeny of said N-plant host and said C-plant host, wherein said progeny comprises said protein.
A method for producing a protein comprising an ExtN and a ExtC, said method comprising:
- (a) transforming an N-plant host with an N-polynucleotide comprising an N-nucleotide sequence that encodes an N-polypeptide comprising said ExtN and an IntN, such that said N-plant host produces said N-polypeptide;
- (b) transforming a C-plant host with a C-polynucleotide comprising a C-nucleotide sequence that encodes a C-polypeptide comprising a IntC and said ExtC, such that said C-plant host produces said C-polypeptide;
- (c) isolating said N-polypeptide from said N-plant host and said C-polypeptide from said C-plant host;
- and
- (d) combining said N-polypeptide and said C-polypeptide in vitro to obtain said protein.
A transgenic plant that produces an active protein comprising an ExtN and a ExtC, wherein said protein is produced from a polynucleotide comprising a nucleotide sequence that encodes said ExtN, said ExtC, and an intein interposed between said ExtN and said ExtC.
A transgenic plant that expresses a polypeptide selected from the group consisting of:
- an ExtN and an IntN;
- and
- an ExtC and an IntC,
- wherein said IntN and said IntC together form an intein, and
- wherein said ExtN and said ExtC together form an active protein.
Dependencies are inferred from the claim text.