Expression of antibodies using single open reading frame (sORF) vector design: Demonstration of manufacturing feasibility.

Efficient production of large quantities of therapeutic antibodies is becoming a major goal of the pharmaceutical industry. We developed a proprietary expression system using a polyprotein precursor-based approach to antibody expression in mammalian cells. In this approach, the coding regions for heavy and light chains are included within a single open reading frame (sORF) separated by an in-frame intein gene.

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.

Mycobacterium tuberculosis prcBA genes encode a gated proteasome with broad oligopeptide specificity.

Genes predicted to be associated with the putative proteasome of Mycobacterium tuberculosis (Mtb) play a critical role in defence of the bacillus against nitrosative stress. However, proteasomes are uncommon in eubacteria and it remains to be established whether Mtb's prcBA genes in fact encode a proteasome. We found that coexpression of recombinant PrcB and PrcA in Escherichia coli over a prolonged period at 37 degrees C allowed formation of an alpha(7)beta(7)beta(7)alpha(7), 750 kDa cylindrical stack of four rings in which all 14 beta-subunits were proteolytically processed to expose the active site threonine.

Structure-function relationships of AE2 regulation by Ca(i)(2+)-sensitive stimulators NH(4+) and hypertonicity.

We showed previously that the nonerythroid anion exchanger AE2 and the erythroid anion exchanger AE1 differ greatly in their regulation by acute changes in intracellular pH (pH(i)) and extracellular pH (pH(o)). We have now examined how AE2, but not AE1, is activated by two stimuli with opposing effects on oocyte pH(i): an alkalinizing stimulus, hypertonicity, and an acidifying stimulus, NH(4)(+). We find that both NH(2)-terminal cytoplasmic and COOH-terminal transmembrane domains of AE2 are required for activation by either stimulus.

Expression and characterization of a synthetic protein C activator in Pichia pastoris.

Protein C activators are proteases that activate protein C in the mammalian coagulation system. A reptilian protein C activator is a critical component in current functional assays for protein C, its cofactor protein S, as well as for the overall status of the protein C pathway. We have constructed a synthetic gene for a protein C activator, based on a published snake-venom polypeptide sequence.