Transport proteins PotD and Crr of Escherichia coli, novel fusion partners for heterologous protein expression.
The Escherichia coli proteome response to the stressor GdnHCl was analyzed through 2-dimensional gel electrophoresis (2-DE). We identified PotD (spermidine/putrescine-binding periplasmic protein) and Crr [glucose-specific phosphotransferase (PTS) enzyme IIA component] as a stress-responsive protein. Even under a stress situation where the total number of soluble proteins decreased by about 10%, 3.5- and 2.2-fold increase was observed in the synthesis of PotD and Crr, respectively. As fusion partners, PotD and Crr dramatically increased the solubility of many aggregation-prone heterologous proteins [e.g. human minipro-insulin (mp-INS), human epidermal growth factor (EGF), human prepro-ghrelin (ppGRN), human interleukin-2(hIL-2), human activation induced cytidine deaminase (AID), human glutamate decarboxylase (GAD(448-585)), Pseudomonas putida cutinase (CUT), human ferritin light chain (hFTN-L), human granulocyte colony-stimulating factor (G-CSF), and cold autoinflammatory syndrome1 protein (NALP3) Nacht domain (NACHT)] in the E. coli cytoplasm. Presumably PotD and Crr were very effective in shielding interactive surfaces of heterologous proteins associated with non-specific protein-protein interactions leading to the formation of inclusion bodies most likely due to intrinsic high folding efficiency, chaperone-like activity, or a combination of both factors. Both the stress-induced proteins were well suited for the production of a biologically active fusion mutant of P. putida cutinase that can be expected to be of biotechnological and commercial interest.
Han
Kyung-Yeon
KY
Department of Chemical and Biological Engineering, Korea University, Anam-Dong 5-1, Sungbuk-Ku, Seoul 136-713, South Korea.
Seo
Hyuk-Seong
HS
Song
Jong-Am
JA
Ahn
Keum-Young
KY
Park
Jin-Seung
JS
Lee
Jeewon
J
D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D002479:Inclusion Bodies / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D033902:Periplasmic Binding Proteins / Q000235:genetics* / Q000378:metabolism; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics* / Q000378:metabolism; D021381:Protein Transport / Q000235:genetics; D020543:Proteome / Q000032:analysis; D016958:Pseudomonas putida / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D033902:Periplasmic Binding Proteins; C099274:PotD protein, E coli; D020543:Proteome; D011993:Recombinant Fusion Proteins; C032242:crr protein, E coli; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D002265:Carboxylic Ester Hydrolases; C019952:cutinase