| abstract |
Useful constructs and methods are disclosed for expressing proteins using primary translation products that are processed in recombinant host cells. Constructs comprising a single open reading frame (sORF) are described for protein expression including expression of multiple polypeptides. Primary translational products (proproteins or polyproteins) contain polypeptides, such as intein or hedgehog family self-processing domains, or variants thereof, inserted in-frame between multiple protein subunits of interest. The primary product may also contain a signal peptide containing a cleavage sequence, such as another proteolytic cleavage or protease recognition site, or a recognition sequence for signal peptidase, separating two or more multiple protein subunits. The sequence of the inserted autoprocessing polypeptide or cleavage site can be engineered to improve the expression efficiency of separate multiple protein subunits. Independent aspects of performing efficient expression, secretion and / or multimeric assembly of proteins such as immunoglobulins are also disclosed. Where the polyprotein contains immunoglobulin heavy and light chain fragments or fragments capable of recognizing antigens, in one embodiment, the selectable stoichiometric ratio is at least two copies of the light chain fragment per heavy chain fragment, and a suitably folded and assembled functional antibody. Is generated. Modified signal peptides are described that include those derived from immunoglobulin light chains. |