Enhanced expression system and methods of use thereof
This disclosure relates to an efficient protein expression system that utilizes piggyBac transposons and/or regulatory elements.
1 . A nucleic acid comprising
a 5′-ITR (inverted terminal repeat) sequence;
a 3′-ITR sequence; and
a regulatory element sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 36, 43, 46, 48, 55, and 58.
2 . The nucleic acid of claim 1 , wherein the 5′ ITR sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 68, and the 3′ ITR sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 60.
3 . The nucleic acid of claim 1 , wherein the 5′ ITR sequence comprises SEQ ID NO: 68 and the 3′ ITR sequence comprises SEQ ID NO: 60.
4 . The nucleic acid of claim 1 , further comprising a 5′-internal domain and a 3′-internal domain, wherein the 5′-internal domain comprise a sequence that is at least 90% identical to SEQ ID NO: 66, wherein the 3′-internal domain comprise a sequence that is at least 90% identical to SEQ ID NO: 67, wherein the 5′-internal domain is immediately adjacent to the 5′-ITR, and the 3′-internal domain is immediately adjacent to the 3′-ITR.
5 . The nucleic acid of claim 1 , wherein the nucleic acid comprises one or more regulatory element sequences selected from the group consisting of SEQ ID NO: 36 and SEQ ID NO: 48.
6 . The nucleic acid of claim 1 , further comprising a promoter and a sequence encoding a polypeptide, wherein the sequence encoding the polypeptide is operably linked to the promoter.
7 . The nucleic acid of claim 6 , wherein the sequence encoding a polypeptide is located between two regulatory element sequences.
8 . The nucleic acid of claim 1 , further comprising a promoter and a sequence encoding two or more polypeptides, wherein the sequence encoding the two or more polypeptides is operably linked to the promoter.
9 . The nucleic acid of claim 8 , wherein the sequence encoding two or more polypeptides encodes an antibody heavy chain and an antibody light chain.
10 . The nucleic acid of claim 1 , wherein in addition to the regulatory element sequence, the nucleic acid further comprises an additional sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 36 and 48.
11 . The nucleic acid of claim 1 , wherein the nucleic acid comprises two or more expression cassettes.
12 . The nucleic acid of claim 1 , wherein the nucleic acid comprises a selection marker.
13 . The nucleic acid of claim 12 , wherein the selection marker is an antibiotic resistance gene, a sequence encoding a fluorescent protein, or lacZ.
14 . The nucleic acid of claim 1 , wherein the 5′-ITR comprises a TR L , a 5′-ITR spacer and an IR L , and the 3′-ITR comprises an IR R , a 3′-ITR spacer and a TR R .
15 . The nucleic acid of claim 14 , wherein the TR L comprises a sequence that is at least 90% identical to a reverse sequence or a reverse complementary sequence of SEQ ID NO: 61; wherein the 5′-ITR spacer comprises a sequence that is at least 90% identical to a reverse sequence or a reverse complementary sequence of SEQ ID NO: 62; wherein the IR L comprises a sequence that is at least 90% identical to a reverse sequence or a reverse complementary sequence of SEQ ID NO: 63; wherein the TR R comprises a sequence that is at least 90% identical to a reverse sequence or a reverse complementary sequence of SEQ ID NO: 64; and wherein the IR R comprises a sequence that is at least 90% identical to a reverse sequence or a reverse complementary sequence of SEQ ID NO: 65.
16 . The nucleic acid of claim 1 , wherein the regulatory element sequence is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 36 and SEQ ID NO: 48.
17 . The nucleic acid of claim 1 , wherein the regulatory element sequence is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 43 and SEQ ID NO: 55.
18 . The nucleic acid of claim 1 , wherein the regulatory element sequence is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 46 and SEQ ID NO: 58.
19 . A vector comprising the nucleic acid of claim 1 .
20 . The vector of claim 19 , comprising the following genetic elements in a 5′ to 3′ direction:
the 5′-ITR comprising a TR L , a 5′-ITR spacer, a IR L ;
a promoter;
the regulatory element sequence;
a protein-coding sequence; and
the 3′-ITR comprising a IR R , a 3′-ITR spacer, a TR R .
21 . The vector of claim 20 , wherein the TR L comprises a sequence that is at least 90% identical to a reverse sequence or a reverse complementary sequence of SEQ ID NO: 61; wherein the 5′-ITR spacer comprises a sequence that is at least 90% identical to a reverse sequence or a reverse complementary sequence of SEQ ID NO: 62; wherein the IR L comprises a sequence that is at least 90% identical to a reverse sequence or a reverse complementary sequence of SEQ ID NO: 63; wherein the TR R comprises a sequence that is at least 90% identical to a reverse sequence or a reverse complementary sequence of SEQ ID NO: 64; and wherein the IR R comprises a sequence that is at least 90% identical to a reverse sequence or a reverse complementary sequence of SEQ ID NO: 65.
22 . A cell comprising the nucleic acid of claim 1 .