Mining mammalian genomes for folding competent proteins using Tat-dependent genetic selection in Escherichia coli.
Recombinant expression of eukaryotic proteins in Escherichia coli is often limited by poor folding and solubility. To address this problem, we employed a recently developed genetic selection for protein folding and solubility based on the bacterial twin-arginine translocation (Tat) pathway to rapidly identify properly folded recombinant proteins or soluble protein domains of mammalian origin. The coding sequences for 29 different mammalian polypeptides were cloned as sandwich fusions between an N-terminal Tat export signal and a C-terminal selectable marker, namely beta-lactamase. Hence, expression of the selectable marker and survival on selective media was linked to Tat export of the target mammalian protein. Since the folding quality control feature of the Tat pathway prevents export of misfolded proteins, only correctly folded fusion proteins reached the periplasm and conferred cell survival. In general, the ability to confer growth was found to relate closely to the solubility profile and molecular weight of the protein, although other features such as number of contiguous hydrophobic amino acids and cysteine content may also be important. These results highlight the capacity of Tat selection to reveal the folding potential of mammalian proteins and protein domains without the need for structural or functional information about the target protein.
Lim
Hyung-Kwon
HK
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, USA.
Mansell
Thomas J
TJ
Linderman
Stephen W
SW
Fisher
Adam C
AC
Dyson
Michael R
MR
DeLisa
Matthew P
MP
D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D016678:Genome; D008322:Mammals / Q000235:genetics*; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames; D017510:Protein Folding; D021381:Protein Transport; D011506:Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D001618:beta-Lactamases / Q000235:genetics
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C408585:twin-arginine translocase complex, E coli; D001618:beta-Lactamases