Abstract: Cell-free synthesis of hydrogen from glucose and cellulosic hydrolysates is provided. Bacterial cells are modified to express high levels of (i) active [FeFe] hydrogenase; (ii) ferredoxin; and (iii) ferredoxin-NADP-reductase (FNR). The cells are then lysed and the lysate is combined with substrate during a production phase, where H2 is produced. The substrate is typically a sugar, e.g. glucose, cellulose hydrolysates, fructose, and the like, including pentose sugars capable of entering the bacterial pentose phosphate cycle. The reaction mixture may be further supplemented with one or more of niacin as a precursor to nicotinamide; a nuclease, particularly a ribonuclease, to break down nucleic acids and generate adenine; and iodoacetamide to inactivate the normal cellular glycolytic pathway and thus maximize conversion yields.

Abstract: Compositions that are EGF polypeptides that possess improved biological activity as compared to the biological activity exhibited by wild-type EGF are provided. Also provided are methods for the preparation of these mutants, methods for the use of these mutants, methods for rationally designing new polypeptide mutants, and methods for screening mutants polypeptides to identify novel EGF mutants with desirable biological activities.

Abstract: The present disclosure relates, in some aspects, to cell-free methods and systems for large-scale conversion of methane to isobutanol, comprising combining, in a bioreactor at elevated pressure, methane, oxygen, and cell lysates containing methane monooxygenase, methanol dehydrogenase, and enzymes that catalyze the conversion of formaldehyde to isobutanol, to form a cell-free reaction mixture, and incubating under suitable conditions the cell-free reaction to convert methane to isobutanol.

Abstract: Compositions of a fusion protein comprising a spatially tethered ferredoxin-NADP-reductase (FNR) and an active [FeFe] hydrogenase, genetic sequences encoding such fusion proteins, and methods of use thereof are provided. The fusion proteins of the invention link an FNR polypeptide to an active [FeFe] hydrogenase through a polypeptide linker. The fusion protein facilitates improved electron transfer through a ferredoxin, and allows direct electron transfer from NADPH to the hydrogenase.

Abstract: A process is described for producing a polypeptide heterologous to E. coli wherein E. coli cells comprising nucleic acid encoding the polypeptide are cultured in a culture medium while feeding to the culture medium a transportable organophosphate, such that the nucleic acid is expressed. The polypeptide is then recovered from the cells.

Abstract: A process is described for producing a polypeptide heterologous to E. coli wherein E. coli cells comprising nucleic acid encoding the polypeptide are cultured in a culture medium while feeding to the culture medium a transportable organophosphate, such that the nucleic acid is expressed. The polypeptide is then recovered from the cells.

Abstract: Compositions that are EGF polypeptides that possess improved biological activity as compared to the biological activity exhibited by wild-type EGF are provided. Also provided are methods for the preparation of these mutants, methods for the use of these mutants, methods for rationally designing new polypeptide mutants, and methods for screening mutants polypeptides to identify novel EGF mutants with desirable biological activities.

Abstract: Methods are provided for the utilization of bacterial cell-free extracts in the synthesis of high yields of virus like particles with encapsidated cargo.

Abstract: Refractile particles containing a heterologous polypeptide as an insoluble aggregate are recovered from bacterial periplasm. The process involves culturing bacterial cells so as to express nucleic acid encoding phage lysozyme and nucleic acid encoding the heterologous polypeptide under separate promoters, disrupting the cells mechanically to release the phage lysozyme so as to release refractile particles from the bacterial cellular matrix, and recovering the released refractile particles from the periplasm. Chloroform is not used in any step and the recovery step minimizes co-recovery of cellular debris with the released refractile particles.

Abstract: Cell-free synthesis of hydrogen from glucose and cellulosic hydrolysates is provided. Bacterial cells are modified to express high levels of (i) active [FeFe] hydrogenase; (ii) ferredoxin; and (iii) ferredoxin-NADP-reductase (FNR). The cells are then lysed and the lysate is combined with substrate during a production phase, where H2 is produced. The substrate is typically a sugar, e.g. glucose, cellulose hydrolysates, fructose, and the like, including pentose sugars capable of entering the bacterial pentose phosphate cycle. The reaction mixture may be further supplemented with one or more of niacin as a precursor to nicotinamide; a nuclease, particularly a ribonuclease, to break down nucleic acids and generate adenine; and iodoacetamide to inactivate the normal cellular glycolytic pathway and thus maximize conversion yields.

Abstract: The embodiments described herein pertain to cells, and methods for preparing cells, that can be used as biocatalysts by altering enzymes that compete for a substrate or product of a pathway of interest such that the targeted enzyme is sensitive to a site-specific protease, which protease is expressed but relocated in the cell to a site where it is not in contact with the targeted enzyme in the intact cell. Upon cell lysis, the protease contacts the target enzyme, which is then inactivated by protease cleavage.

Abstract: Genetically manipulated cells, lysates of such cells, systems, and methods of use thereof are provided, where one or more enzymes in a pathway of interest are genetically modified to incorporate a peptide sequence that provides for relocation of the protein, e.g., to the periplasm, so as to sequester the enzyme, and where the enzyme controls flux in the pathway of interest.

Abstract: A process is described for producing a polypeptide heterologous to E. coli wherein E. coli cells comprising nucleic acid encoding the polypeptide are cultured in a culture medium while feeding to the culture medium a transportable organophosphate, such that the nucleic acid is expressed. The polypeptide is then recovered from the cells.

Abstract: Refractile particles containing a heterologous polypeptide as an insoluble aggregate are recovered from bacterial periplasm. The process involves culturing bacterial cells so as to express nucleic acid encoding phage lysozyme and nucleic acid encoding the heterologous polypeptide under separate promoters, disrupting the cells mechanically to release the phage lysozyme so as to release refractile particles from the bacterial cellular matrix, and recovering the released refractile particles from the periplasm. Chloroform is not used in any step and the recovery step minimizes co-recovery of cellular debris with the released refractile particles.

Abstract: Refractile particles containing a heterologous polypeptide as an insoluble aggregate are recovered from bacterial periplasm. The process involves culturing bacterial cells so as to express nucleic acid encoding phage lysozyme and nucleic acid encoding the heterologous polypeptide under separate promoters, disrupting the cells mechanically to release the phage lysozyme so as to release refractile particles from the bacterial cellular matrix, and recovering the released refractile particles from the periplasm. Chloroform is not used in any step and the recovery step minimizes co-recovery of cellular debris with the released refractile particles.

Abstract: Compositions and methods are provided for the control of direct protein attachment to virus like particles where virus structural proteins that have been modified to comprise an unnatural amino acid at a pre-determined site are reacted with one or more “display” polypeptides that also comprise an unnatural amino acid at a pre-determined site in a one step reaction. The compositions of the invention are useful for various purposes where it is desirable to efficiently and directly attach multiple polypeptides to a single carrier entity, particularly where two or more different polypeptides are attached to a single carrier.

Abstract: Methods are provided for the utilization of bacterial cell-free extracts in the synthesis of high yields of virus like particles with encapsidated cargo.

Abstract: Biological macromolecules are synthesized in vitro in a thin film that provides for high surface area/volume ratio, allowing improved yield in scaled up reactions.

Abstract: A process is described for producing a polypeptide heterologous to E. coli wherein E. coli cells comprising nucleic acid encoding the polypeptide are cultured in a culture medium while feeding to the culture medium a transportable organophosphate, such that the nucleic acid is expressed. The polypeptide is then recovered from the cells.

Abstract: Compositions and methods are provided for the enhanced in vitro synthesis of polypeptides. In order to improve the performance of in vitro protein synthesis reactions, metabolic inhibitors, or manipulation of a source organism, is used to diminish or avoid the action of enzymes responsible for undesirable amino acids production or depletion. A homeostatic system may be used for production of ATP, where the required high energy phosphate bonds are generated in situ, e.g. through coupling with an oxidation reaction. The homeostatic energy source will typically lack high energy phosphate bonds itself, and will therefore utilize free phosphate in the reaction mix during generation of ATP. The homeostatic energy source is provided in combination with an enzyme that catalyzes the creation of high energy phosphate bonds and with an enzyme that can use that high energy phosphate bond to regenerate ATP.