US 12,649,931 B2Granted

SmartBac baculovirus expression system and application thereof

AssigneeInstitute of Biophysics, Chinese Pie Academy of Sciences

InventorsYujia Zhai, Fei Sun

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Abstract

The present invention discloses a SmartBac baculovirus expression system and application thereof. The system can comprise a acceptor plasmid (containing fragment A or fragments B and C) and a donor plasmid (containing fragment D); the fragment A contains a promoter, a sequence encoding a protease, a protease cleavage site, an insertion region of a gene encoding a target object to be expressed and a termination sequence; the fragment B contains a promoter, a sequence encoding a protease and a termination sequence; the fragment C contains a promoter, an insertion region of a gene encoding a target object to be expressed and a termination sequence; the fragment D contains a promoter, an insertion region of a gene encoding a target object to be expressed and a termination sequence. The present invention also provides three cloning strategies to achieve the expression of protein complexes with molecular weights of less than 600 kDa and the expression of protein complexes with molecular weights of no less than 600 kDa and efficient screening of a subunit most suitable for adding a purification tag. The present invention is of great significance for recombinantly expressing protein complexes with complex components and large molecular weights in insect cells.

A SmartBac baculovirus expression system, which is shown as follows:

SmartBac baculovirus expression system A, comprising an acceptor plasmid component and a donor plasmid component;
wherein the acceptor plasmid component and the donor plasmid component are capable of being recombined and fused into one plasmid component;
the acceptor plasmid component is the acceptor plasmid A and/or the acceptor plasmid B;
the acceptor plasmid A comprises a DNA fragment A comprising, in order from upstream to downstream,
a promoter A,
a gene sequence encoding a protease,
a nucleotide sequence encoding a recognition sequence for a cleavage site of the protease,
an insertion region of a gene encoding a target object to be expressed, and
a termination sequence A;
the acceptor plasmid B comprises a DNA fragment B comprising, in order from upstream to downstream,
a promoter B,
a gene sequence encoding the protease, and
a termination sequence B;
and
a DNA fragment C comprising, from upstream to downstream,
a promoter C,
an insertion region of a gene encoding a target object to be expressed, and
a termination sequence C;
the donor plasmid component contains a DNA fragment D comprising, from upstream to downstream,
a promoter D,
an insertion region of a gene encoding a target object to be expressed, and
a termination sequence D;
wherein
the target object is selected from the group consisting of proteins, protein subunits, protein fragments, polypeptides, and polypeptide fragments wherein in the SmartBac baculovirus expression system A, the sequence of the DNA fragment A is set forth in positions 1235-3971 of SEQ ID NO:
1 or positions 1235-3971 of SEQ ID NO:
2;
wherein in the SmartBac baculovirus expression system A, the sequence of the DNA fragment B is set forth in positions 1208-3252 of SEQ ID NO:
3 or positions 1208-3249 of SEQ ID NO:
4;
wherein in the SmartBac baculovirus expression system A, the sequence of the DNA fragment C is set forth in positions 3306-4521 of SEQ ID NO:
3 or positions 3303-4518 of SEQ ID NO:
4;
or
wherein in the SmartBac baculovirus expression system A, the sequence of the DNA fragment D is set forth in positions 259-3188 of SEQ ID NO:
5 or positions 259-3188 of SEQ ID NO:
6.

33 more elements

Abstract

Claims15