Soluble expression of recombinant olive flounder hepcidin I using a novel secretion enhancer.
Expression of olive flounder hepcidin I (HepI) fused with truncated OmpA signal peptides (OmpASP(tr)) as directional signals does not produce soluble fusion proteins. However, by inserting amino acid segments (xxx) varying in pI and hydrophobicity/hydrophilicity into a leader sequence containing a truncated OmpASP (OmpASP(tr)) and a factor Xa cleavage site (Xa) [OmpASP(tr)|(xxx)|Xa], we were able in some cases to express soluble recombinant HepI. Soluble expression of the recombinant protein strongly correlated with (xxx) insertions of high pI and hydrophilicity. Therefore, we modified the OmpASP(tr)|(xxx)|Xa sequence by inserting Arg and Lys into (xxx) to increase the hydrophilicity of the signal peptide region. These modifications enhanced the expression of soluble recombinant HepI. Hydropathic profile analysis of the OmpASP(tr)|(xxx)|Xa| HepI fusion proteins revealed that the transmembrane-like domains derived from the OmpASP(tr)|(xxx)|Xa sequence were larger than the internal positively charged domain native to HepI. It should therefore be possible to overcome the obstacle of internal positively charged domains to obtain soluble expression of recombinant proteins by monitoring the hydrophilicity and hydropathic profile of the signal peptide region using a computer program.
Lee
Sang Jun
SJ
Biotechnology Research Institute, National Fisheries Research and Development Institute, Pusan 619-902, Korea. sjlee@nfrdi.re.kr
Park
In Suk
IS
Han
Yun Hee
YH
Kim
Young Ok
YO
Reeves
Peter R
PR
D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis / Q000235:genetics*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D005432:Flounder / Q000235:genetics; D064451:Hepcidins; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012995:Solubility
D023181:Antimicrobial Cationic Peptides; D001425:Bacterial Outer Membrane Proteins; D064451:Hepcidins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C078167:OMPA outer membrane proteins