title: N-terminal pI determines the solubility of a recombinant protein lacking an internal transmembrane-like domain in E. coli.
abstract: 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.
authors.affiliation: Biotechnology Research Division, National Fisheries Research and Development Institute, Pusan 619-902, Korea. sangjl@nfrdi.go.kr
authors.forename: Sang Jun
authors.initials: SJ
authors.lastname: Lee
authors.forename: Yun Hee
authors.initials: YH
authors.lastname: Han
authors.forename: Young Ok
authors.initials: YO
authors.lastname: Kim
authors.forename: Bo Hye
authors.initials: BH
authors.lastname: Nam
authors.forename: Hee Jeong
authors.initials: HJ
authors.lastname: Kong
authors.forename: Kyung Kil
authors.initials: KK
authors.lastname: Kim
mesh_terms: 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
chemical_list: D003062:Codon; D011994:Recombinant Proteins