US 12,227,778 B2
Cell-free expression system having novel inorganic polyphosphate-based energy regeneration
Alexander Koglin, Santa Fe, NM (US); and Michael Humbert, Santa Fe, NM (US)
Assigned to NATURE'S TOOLBOX, INC., Rio Rancho, NM (US)
Filed by NTxBio, LLC, Santa Fe, NM (US)
Filed on Sep. 30, 2020, as Appl. No. 17/039,575.
Application 17/039,575 is a continuation in part of application No. 16/309,074, granted, now 11,136,586, previously published as PCT/US2018/012121, filed on Jan. 2, 2018.
Claims priority of provisional application 62/440,975, filed on Dec. 30, 2016.
Prior Publication US 2021/0024912 A1, Jan. 28, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/52 (2006.01); C12N 9/04 (2006.01); C12N 9/12 (2006.01)
CPC C12N 9/52 (2013.01) [C12N 9/0006 (2013.01); C12N 9/1205 (2013.01); C12N 9/1229 (2013.01); C12N 9/1247 (2013.01); C12Y 101/01014 (2013.01); C12Y 207/0102 (2013.01); C12Y 207/04001 (2013.01); C12Y 207/07006 (2013.01); C12Y 304/24068 (2013.01)] 30 Claims
 
1. A cell-free protein expression system comprising:
a bioreactor having:
a reaction mixture containing isolated cell-free reaction components necessary for in vitro macromolecule synthesis;
at least one isolated nucleic acid synthesis template; and
a cellular adenosine triphosphate (ATP) energy regeneration system comprising:
purified Adenosyl Kinase (AdK) enzyme according to SEQ ID NO: 8, or fragment thereof, or an Adk enzyme having a sequence having at least 95% sequence homology with SEQ ID NO: 8, that catalyzes the conversion of adenosine diphosphate into adenosine triphosphate (ATP) and adenosine monophosphate (AMP);
purified Polyphosphate Kinase (PPK) enzyme according to SEQ ID NO: 11, or fragment thereof, or a PPK enzyme having a sequence having at least 95% sequence homology with SEQ ID NO: 11, that catalyzes the reversible formation of ATP from ADP and inorganic polyphosphate (PPi);
isolated PPi; and
isolated adenosine monophosphate (AMP);
wherein said AdK and PPK enzymes work synergistically to regenerate cellular ATP energy from PPi and AMP.