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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. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [ABSTRACT FROM AUTHOR]

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°C allowed formation of an α7β7β7α7, 750 kDa cylindrical stack of four rings in which all 14 β-subunits were proteolytically processed to expose the active site threonine. In contrast to another Actinomycete, Rhodococcus erythropolis, Mtb's β-chain propeptide was not required for particle assembly. Peptidolytic activity of the 750 kDa particle towards a hydrophobic oligopeptide was nearly two orders of magnitude less than that of the Rhodococcus 20S proteasome, and unlike eukaryotic and archaeal proteasomes, activity of the Mtb 750 kDa particle could not be stimulated by SDS, Mg2+ or Ca2+. Electron microscopy revealed what appeared to be obstructed α-rings in the Mtb 750 kDa particle. Deletion of the N-terminal octapeptide from Mtb's α-chain led to disappearance of the apparent obstruction and a marked increase of peptidolytic activity. Unlike proteasomes isolated from other Actinomycetes, the open-gate Mtb mutant 750 kDa particle cleaved oligopeptides not only after hydrophobic residues but also after basic, acidic and small, neutral amino acids. Thus, Mtb encodes a broadly active, gated proteasome that may work in concert with an endogenous activator. [ABSTRACT FROM AUTHOR]