| APC Biomaterials, LLC. |
The T2 Bioprocess Platform: The T2 bioprocess platform synthesizes a recombinant peptide or small protein as a
C-terminal extension of an optimized UBL (ubiquitin-like protein), called T2, and then employs a UBL-specific protease
to release the peptide. T2 and its cognate proteases have been optimized for maximum expression level, purification,
stability and in vitro cleavage kinetics. Both were engineered with histidine tags to facilitate initial purification of the T2
fusion protein using immobilized metal chelation chromatography and subsequent removal of the T2 and protease from
the reaction mixture. The specificity of the protease is exquisite. No exopeptidase activity has ever been observed
and the peptide products are recovered intact. Our recombinant UBL protease is also extremely robust. Cleavage
reactions typically approach 100% completion in a few hours at 37ºC, even when the molar ratio of protease to
substrate is 1:102-106. T2 itself is resistant to degradation by bacterial proteases and can accumulate to nearly 90% of
the total intracellular protein. Preliminary studies and cost projections suggest that peptides ranging from about 15 to
70 amino acids can be produced for dollars per gram, in contrast to chemical synthetic methods for which costs and
lead times dramatically increase with the length of the peptide product. Chemical synthesis produces many closely
related impurities which are expensive to remove, a problem that our method avoids. An additional advantage of the
T2 platform over chemical synthesis is the lack of significant organic waste, which also contributes to cost reduction
and is more environmentally-friendly. Another major advantage is the rapidity with which each product can be
generated and the relatively generic nature of the process for many peptide and protein products, facilitating scale-up.
C-terminal extension of an optimized UBL (ubiquitin-like protein), called T2, and then employs a UBL-specific protease
to release the peptide. T2 and its cognate proteases have been optimized for maximum expression level, purification,
stability and in vitro cleavage kinetics. Both were engineered with histidine tags to facilitate initial purification of the T2
fusion protein using immobilized metal chelation chromatography and subsequent removal of the T2 and protease from
the reaction mixture. The specificity of the protease is exquisite. No exopeptidase activity has ever been observed
and the peptide products are recovered intact. Our recombinant UBL protease is also extremely robust. Cleavage
reactions typically approach 100% completion in a few hours at 37ºC, even when the molar ratio of protease to
substrate is 1:102-106. T2 itself is resistant to degradation by bacterial proteases and can accumulate to nearly 90% of
the total intracellular protein. Preliminary studies and cost projections suggest that peptides ranging from about 15 to
70 amino acids can be produced for dollars per gram, in contrast to chemical synthetic methods for which costs and
lead times dramatically increase with the length of the peptide product. Chemical synthesis produces many closely
related impurities which are expensive to remove, a problem that our method avoids. An additional advantage of the
T2 platform over chemical synthesis is the lack of significant organic waste, which also contributes to cost reduction
and is more environmentally-friendly. Another major advantage is the rapidity with which each product can be
generated and the relatively generic nature of the process for many peptide and protein products, facilitating scale-up.
- Generic purification scheme streamlines
downstream process development - Process scaleable to larger quantities
- Rapid turnaround (4-8 weeks)
- Often overcomes toxicity of peptide to host cells
enabling expression - Expression in bacteria; superior yields
- No residues added to peptide product
- Yield increases with size, opposite of chemical
synthesis - Superior stability
- Green alternative to chemical peptide synthesis
- Economical and efficient for synthesis of large or
complex peptides (>15 aa); milligram quantities &
up - Purity > 95%, suitable for SAR & animal work
- Removable purification tag overcomes
purification barriers
| Protease reactions typically complete in 2 hours using enzyme:substrate ratios of 1:102 - 106 |
- Relevant applications:
- Custom peptide synthesis
- Peptide drugs
- Subunit vaccines
- Recombinant peptide libraries
- Custom immunogens for antibody synthesis
- Diagnostic analytes & calibrators
- Screening tools
- Peptide drug optimization; SAR studies
- Bioprocess development
- Scale-up peptide production process
References
- Pilon, A.L., Yost, P., Chase, T.E., Lohnas, G.L., Burkett, T., Roberts, S.R., and W.E. Bentley.
Ubiquitin Fusion Technology: Bioprocessing of Peptides. Biotech. Prog. 13:374-379 (1997). - Pilon, A.L., Yost, P., Chase, T.E., Lohnas, G.L., and W.E. Bentley. High-level expression and
efficient recovery of ubiquitin fusion proteins from Escherichia coli. Biotechnology
Progress 12:331-337 (1996).
| Custom recombinant peptide synthesis |