Efficient and scalable method for scaling up cell free protein synthesis in batch mode.
A novel method for general cell free system scale-up in batch mode was applied to expression of E. coli chloramphenicol acetyl transferase (CAT) and a GMCSF-scFv fusion protein being developed as a B-cell lymphoma vaccine candidate (GLH). Performance of two different E. coli based cell-free systems was evaluated using the new scale-up approach. Reaction volumes from 15 to 500 microL were tested for both products and both reaction systems. In each case, the new scale-up method preserved total, soluble, and active volumetric yields of GLH and CAT at every reaction volume. At the 500 microL reaction volume, the PANOx SP system produced 560 +/- 36 microg/mL of active CAT and 99 +/- 10 microg/mL of active GLH protein using the new thin film approach whereas 500 microL test tube reactions produced 250 +/- 42 microg/mL and 72 +/- 7 microg/mL of active CAT and GLH respectively. Similarly, 500 microL cell-free synthesis reactions with the Cytomim system produced 481 +/- 38 microg/mL of active CAT and 109 +/- 15 microg/mL active GLH respectively in thin films compared to 29 +/- 7 microg/mL of active CAT and 5 +/- 2 microg/mL of active GLH protein in 500 microL test tube reactions. The new thin film approach improves oxygen supply for the Cytomim system, and increases the availability of hydrophobic surfaces. Analysis suggests that these surfaces provide significant benefit for protein expression and folding. We believe that this approach provides a general reaction scale-up technology that will be suitable for any protein target, cell free system, and reaction volume.
Voloshin
Alexei M
AM
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Swartz
James R
JR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis; D019149:Bioreactors; D019496:Cancer Vaccines / Q000032:analysis / Q000096:biosynthesis; D002474:Cell-Free System; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004867:Equipment Design; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000032:analysis / Q000096:biosynthesis; D007218:Industrial Microbiology / Q000379:methods*; D051379:Mice; D014176:Protein Biosynthesis; D013997:Time Factors
D000911:Antibodies, Monoclonal; D019496:Cancer Vaccines; D029968:Escherichia coli Proteins; D015500:Chloramphenicol O-Acetyltransferase