Synthesis of proteins by cell-free protein expression

A method for producing protein or biological macromolecule from a nucleic acid template in an in vitro protein or biological macromolecule synthesis system comprising:

• contacting said nucleic acid template with one or more energy sources providing chemical energy for protein or biological macromolecule synthesis to form a mixture wherein at least one of said energy sources is a glycolytic intermediate;
• contacting said nucleic acid template and said one or more energy sources with a cell extract;
• and
• incubating said mixture under conditions sufficient to produce at least one protein or biological macromolecule encoded by said template.

A composition for in vitro protein or biological, macromolecule synthesis comprising:

• one or more energy sources providing chemical energy for protein or biological macromolecule synthesis wherein at least one of said energy sources is a glycolytic intermediate;
• further comprising components selected from the group consisting of:
• at least one extract from a cell having reduced activity of at least one enzyme that catalyzes hydrolysis of high-energy phosphate bonds or hydrolysis or formation of phosphodiester bonds;
• at least one inhibitor of at least one enzyme that catalyzes hydrolysis of high-energy phosphate bonds or hydrolysis or formation of phosphodiester bonds;
• at least one inhibitor of an enzyme that degrades said template;
• and
• at least one extract from a cell having reduced degradative effect on said template.