Biomedical subjects

James R Swartz

Publications and source records attributed to James R Swartz.

21 records · Page 2Linked to original sources

PubMedDate not supplied

Expression of active murine granulocyte-macrophage colony-stimulating factor in an Escherichia coli cell-free system.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important cytokine in the mammalian immune system. It has been expressed in Escherichia coli with the same biological activity as the native protein. Here, we report the synthesis of a murine recombinant GM-CSF in an E. coli cell-free protein synthesis system with a high yield. Since there are two disulfide bonds in the native structure of GM-CSF, an oxidizing redox potential of the reaction mixture was required. By pretreating the cell extract with iodoacetamide (IAM), the reducing activity of the cell extract was inactivated, and upon further application of an oxidized glutathione buffer, most of the synthesized GM-CSF was found in its oxidized form. However, the GM-CSF thus formed showed low activity because of poor folding. With the addition of DsbC, the periplasmic disulfide isomerase from E. coli, a high yield of active GM-CSF was produced in the cell-free reaction. Finally, successful folding of the cell-free synthesized GM-CSF-his6 was confirmed by its cell-proliferation activity after purification with a Ni2+ chelating column.

Animals↗

PubMedDate not supplied

Streamlining Escherichia coli S30 extract preparation for economical cell-free protein synthesis.

Escherichia coli extracts activate cell-free protein synthesis systems by providing the catalysts for translation and other supporting reactions. Recent results suggest that high-density fermentations can be used to provide the source cells, but the subsequent cell extract preparation procedure requires multiple centrifugation and dialysis steps as well as an expensive runoff reaction. In the work reported here, the extract preparation protocol duration was reduced by nearly 50% by significantly shortening several steps. In addition, by optimizing the runoff incubation, overall reagent costs were reduced by 70%. Nonetheless, extracts produced from the shorter, less expensive procedure were equally active. Crucial steps were further examined to indicate minimal ribosome loss during the standard 30,000g centrifugations. Furthermore, sucrose density centrifugation analysis indicated that although an incubation step significantly activates the extract, ribosome/polysome dissociation is not required. These insights suggest that consistent cell extract can be produced more quickly and with considerably less expense for large-scale cell-free protein production, especially when combined with high-density fermentation protocols.

Cell-Free System↗

PubMedDate not supplied

An economical method for cell-free protein synthesis using glucose and nucleoside monophosphates.

Cell-free protein synthesis reactions have not been seriously considered as a viable method for commercial protein production mainly because of high reagent costs and a lack of scalable technologies. Here we address the first issue by presenting a cell-free protein synthesis system with comparable protein yields that removes the most expensive substrates and lowers the cell-free reagent cost by over 75% (excluding extract, polymerase, and plasmid) while maintaining high energy levels. This system uses glucose as the energy source and nucleoside monophosphates (NMPs) in place of nucleoside triphosphates (NTPs) as the nucleotide source. High levels of nucleoside triphosphates are generated from the monophosphates within 20 min, and the subsequent energy charge is similar in reactions beginning with either NTPs or NMPs. Furthermore, significant levels (>0.2 mM) of all NTPs are still available at the end of a 3-h incubation, and the total nucleotide pool is stable throughout the reaction. The glucose/NMP reaction was scaled up to milliliter scale using a thin film approach. Significant yields of active protein were observed for two proteins of vastly different size: chloramphenicol acetyl transferase (CAT, 25 kDa) and beta-galactosidase (472 kDa). The glucose/NMP cell-free reaction system dramatically reduces reagent costs while supplying high protein yields.

Biochemistry↗