N-terminal pI determines the solubility of a recombinant protein lacking an internal transmembrane-like domain in E. coli.
We examined whether the isoelectric point (pI) of the Nterminal region of the recombinant protein 7xMefp1 acts as a universal index for expression of the protein in soluble form in E. coli. Expression analysis of 7xMefp1 fused to various N-terminal sequences with pI values ranging from 2.73 to 13.35 yielded three pI range-specific curves (acidic, neutral, and alkaline curves at pI 2.73-3.25, 4.61-9.58, and 9.90-13.35, respectively) for soluble expression (by facilitated diffusion) as a proportion of total protein. For neutral N-termini (pI 4.61-9.58), the total amount of rMefp1 expressed was minimally affected by DeltaG(RNA) for unfolding the mRNA secondary structure. The highly hydrophilic nature of longer N-terminal sequences with strongly acidic and alkaline pI values reduced the translation of rMefp1-encoding transcripts, thereby reducing the amount of soluble rMefp1 produced. After characterizing both feedback and non-feedback regulation in the acidic, alkaline, and neutral pI ranges, we suggest that three different pI range-specific soluble expression curves exist for the recombinant protein, each defined by specific ranges of the leader sequence pI values.
Lee
Sang Jun
SJ
Biotechnology Research Division, National Fisheries Research and Development Institute, Pusan 619-902, Korea. sangjl@nfrdi.go.kr
Han
Yun Hee
YH
Kim
Young Ok
YO
Nam
Bo Hye
BH
Kong
Hee Jeong
HJ
Kim
Kyung Kil
KK
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry*; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism*; D020440:Gene Duplication / Q000235:genetics; D007526:Isoelectric Point; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism*; D012995:Solubility
D003062:Codon; D011994:Recombinant Proteins