title: Stability of a guest protein depends on stability of a host protein in insertional fusion.
abstract: Insertional fusion between host and guest protein domains has been employed to create multi-domain protein complexes displaying integrated and coupled functionalities. The effects of insertional fusion on the stability of a guest protein are however rather controversial. In the study described here, we examined whether the stability of inserted TEM1 beta-lactamase (BLA), as a guest protein, might be affected by the stability of a maltodextrin-binding protein (MBP), as a host protein. Our results indicate that expression levels and in vitro stability of the BLA domain were significantly higher when inserted into thermophilic MBP from Pyrococcus furiosus (PfMBP) compared to mesophilic MBP from Escherichia coli (EcMBP). Moreover, insertion into PfMBP at selected sites was found to improve thermal stability of the BLA domain without compromise in expression levels and BLA activity. Kinetic stabilization during prolonged thermal denaturation of the BLA domain was not guaranteed by insertion into PfMBP, but rather relied on the insertion sites. Taken together, we provide evidence that (i) the stability of the guest protein depended on the stability of the host protein in insertional fusion and (ii) insertion into PfMBP, at least at selected locations, can serve as a novel method of improving protein thermal stability.
authors.affiliation: Othmer-Jacobs Department of Chemical and Biological Engineering, Polytechnic Institute of New York University, Brooklyn, New York 11201, USA.
authors.forename: Brennal
authors.initials: B
authors.lastname: Pierre
authors.forename: Tina
authors.initials: T
authors.lastname: Xiong
authors.forename: Leonie
authors.initials: L
authors.lastname: Hayles
authors.forename: Visweswara Reddy
authors.initials: VR
authors.lastname: Guntaka
authors.forename: Jin Ryoun
authors.initials: JR
authors.lastname: Kim
mesh_terms: D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology; D008958:Models, Molecular; D020027:Pyrococcus furiosus / Q000737:chemistry / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D001618:beta-Lactamases