• Molecules and Cells
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• 제26권1호
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• pp.34-40
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• 2008

To express an increased level of recombinant Mefp1 (marine mussel adhesive protein) in soluble form, we constructed expression vectors encoding truncated OmpA signal peptide-Mefp1 fusion proteins. OmpA signal peptide (OmpASP) is the 21 residue peptide fragment of the 23 residue OmpA signal sequence cleavable by signal peptidase I. We successfully produced increased levels of soluble recombinant Mefp1 (rMefp1) with various deletions of OmpASP, and found that the increased expression was caused by the increased pI of the N-terminus of the fusion proteins (${\geq}10.55$). All the OmpA signal peptide segments of 3-21 amino acids in length had the same pI value (10.55). Our results suggest that the pI value of the truncated OmpASP ($OmpASP_{tr}$) play an important role in directional signaling for the fusion protein, but we found no evidence for the presence of a secretion enhancer in OmpASP. For practical applications, we increased the expression of soluble rMefp1 with $OmpASP_{tr}$ peptides as directional signals, and obtained rMefp1 with the native amino terminus (nN-rMefp1) using an $OmpASP_{tr}$ Xa leader sequence that contains the recognition site for Xa protease.

• Molecules and Cells
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• 제26권2호
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• pp.140-145
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• 2008

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) [$OmpASP_{tr}{\mid}(xxx){\mid}Xa$], 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 $OmpASP_{tr}{\mid}(xxx){\mid}Xa$ 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 $OmpASP_{tr}{\mid}(xxx){\mid}Xa$ HepI fusion proteins revealed that the transmembrane-like domains derived from the $OmpASP_{tr}{\mid}(xxx){\mid}Xa$ 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.