Novel GFP expression using a short N-terminal polypeptide through the defined twin-arginine translocation (Tat) pathway.
Escherichia coli is frequently used as a convenient host organism for soluble recombinant protein expression. However, additional strategies are needed for proteins with complex folding characteristics. Here, we suggested that the acidic, neutral, and alkaline isoelectric point (pI) range curves correspond to the channels of the E. coli type-II cytoplasmic membrane translocation (periplasmic translocation) pathways of twin-arginine translocation (Tat), Yid, and general secretory pathway (Sec), respectively, for unfolded and folded target proteins by examining the characteristic pI values of the N-termini of the signal sequences or the leader sequences, matching with the known diameter of the translocation channels, and analyzing the N-terminal pI value of the signal sequences of the Tat substrates. To confirm these proposed translocation pathways, we investigated the soluble expression of the folded green fluorescent protein (GFP) with short N-terminal polypeptides exhibiting pI and hydrophilicity separately or collectively. This, in turn, revealed the existence of an anchor function with a specific directionality based on the N-terminal pI value (termed as N-terminal pI-specific directionality) and distinguished the presence of the E. coli type-II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for the unfolded and folded target proteins. We concluded that the pI value and hydrophilicity of the short N-terminal polypeptide, and the total translational efficiency of the target proteins based on the ΔGRNA value of the N-terminal coding regions are important factors for promoting more efficient translocation (secretion) through the largest diameter of the Tat channel. These results show that the short N-terminal polypeptide could substitute for the Tat signal sequence with improved efficiency.
Biotechnology Research Division, National Fisheries Research and Development Institute, Busan 619-902, Korea. sangjl@nfrdi.go.kr
Sang Jun
SJ
Lee
Yun Hee
YH
Han
Young Ok
YO
Kim
Bo Hye
BH
Nam
Hee Jeong
HJ
Kong
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D019295:Computational Biology; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000235:genetics*; D055550:Protein Stability; D021381:Protein Transport; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D055571:Secretory Pathway / Q000235:genetics; D019076:Transgenes / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; D000069816:SEC Translocation Channels; C412443:YIDC protein, E coli; C408585:twin-arginine translocase complex, E coli; D049452:Green Fluorescent Proteins; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins