Compositions and methods for enhanced expression of recombinant polypeptides from a single vector using a peptide cleavage site
View Patent ↗Vector constructs for expression of two or more functional proteins or polypeptides under operative control of a single promoter and methods of making and using the same are described. The vectors comprise a self-processing cleavage site between each respective protein or polypeptide coding sequence. The vector constructs include the coding sequence for a self-processing cleavage site and may further include an additional proteolytic cleavage sequence which provides a means to remove the self processing peptide sequence from expressed protein(s) or polypeptide(s). The vector constructs find utility in methods for enhanced production of biologically active proteins and polypeptides in vitro and in vivo.
1. A method for producing an immunoglobulin or a fragment of the immunoglobulin having antigen-binding activity, wherein the immunoglobulin fragment is selected from the group consisting of Fab, F(ab′) 2 , Fv(scFv) and Fv immunoglobulin fragments, comprising the steps of:
(i) transducing a host cell with a vector comprising in the 5′ to 3′ direction, a cytomegalovirus enhancer/chicken beta-actin (CAG) promoter operably linked to all of (1) a coding sequence for a heavy chain of the immunoglobulin or a fragment of the heavy chain, (2) a coding sequence for a furin cleavage site, (3) a coding sequence for a 2A self-processing cleavage site and (4) a coding sequence for a light chain of the immunoglobulin or a fragment of the light chain; and
(ii) expressing the heavy chain of the immunoglobulin or the fragment of the heavy chain and the light chain of the immunoglobulin or the fragment of the light chain in the transduced host cell, wherein the heavy chain of the immunoglobulin or the fragment of the heavy chain and the light chain of the immunoglobulin or the fragment of the light chain are expressed in an equimolar or close to equimolar ratio.
2. The method according to claim 1 , wherein the 2A self-processing cleavage site is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.
3. The method according to claim 2 , wherein the 2A self-processing cleavage site is SEQ ID NO:6.
4. The method according to claim 3 , wherein the furin cleavage site has the consensus sequence SEQ ID NO:10.
5. The method according to claim 1 , wherein the coding sequence for the 2A self-processing cleavage site is SEQ ID NO:14.
6. The method according to claim 1 , wherein the furin cleavage site has the consensus sequence SEQ ID NO:10.
7. The method according to claim 1 , wherein the vector is an adeno-associated virus (AAV) vector.