Expression of antibodies using single-open reading frame vector design and polyprotein processing from mammalian cells.
We describe a novel polyprotein precursor-based approach to express antibodies from mammalian cells. Rather than expressing heavy and light chain proteins from separate expression units, the antibody heavy and light chains are contained in one single-open reading frame (sORF) separated by an intein gene fused in frame. Inside mammalian cells this ORF is transcribed into a single mRNA, and translated into one polypeptide. The antibody heavy and light chains are separated posttranslationally, assembled into the functional antibody molecule, and secreted into culture medium. It is demonstrated that Pol I intein from P. horikoshii mediates protein splicing and cleavage reactions in mammalian cells, in the context of antibody heavy and light chain amino acid sequences. To allow the separation of antibody heavy chain, light chain, and the intein, we investigated a number of intein mutations designed to inhibit intein-mediated splicing but preserve cleavage reactions. We have also designed constructs in which the signal peptide downstream from intein has altered hydrophobicity. The use of some of these mutant constructs resulted in more efficient antibody secretion, highlighting areas that can be further explored in improving such an expression system. An antibody secreted using one of the sORF constructs was characterized. This antibody has correct N-terminal sequences for both of its heavy and light chains, correct heavy and light chain MW as well as intact MW as measured by mass spectrometry. Its affinity to antigen, as measured by surface plasmon resonance (SPR), is indistinguishable from that of the same antibody produced using conventional method.
Abbott Bioresearch Center, 100 Research Drive, Worcester, MA 01605, USA. yune.kunes@abbott.com
Yune Z
YZ
Kunes
Wendy R
WR
Gion
Emma
E
Fung
Jochen G
JG
Salfeld
Rong-Rong
RR
Zhu
Paul
P
Sakorafas
Gerald R
GR
Carson
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047668:Inteins; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D020815:Polyproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011499:Protein Processing, Post-Translational; D043902:Pyrococcus horikoshii / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D019843:Archaeal Proteins; D020815:Polyproteins; D011993:Recombinant Fusion Proteins; D004256:DNA Polymerase I