Heterologous gene expression using self-assembled supra-molecules with high affinity for HSP70 chaperone

被引:38
作者
Ahn, JY
Choi, H
Kim, YH
Han, KY
Park, JS
Han, SS
Lee, J
机构
[1] Korea Univ, Dept Chem & Biol Engn, Seoul 136713, South Korea
[2] Korea Univ, Sch Life Sci & Biotechnol, Seoul 136713, South Korea
关键词
D O I
10.1093/nar/gki692
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Contrary to the results of direct expression, various human proteins (ferritin light-chain, epithermal growth factor, interleukin-2, prepro-ghrelin, deletion mutants of glutamate decarboxylase and arginine deiminase, and mini-proinsulin) were all soluble in Escherichia coli cytoplasm when expressed with the N-terminus fusion of ferritin heavy-chain (FTN-H). Through systematic investigations, we have found that a specific peptide motif within FTN-H has a high affinity to HSP70 chaperone DnaK, and that the peptide motif was composed of a hydrophobic core of three residues (Ile, Phe and Leu) and two flanking regions enriched with polar residues (Gly, Gln and Arg). It was also observed that all the recombinant proteins expressed with the fusion of FTN-H formed spherical nanoparticles with diameters of 10-15 nm, as confirmed by the transmission electron microscopy image. The protein nanoparticles are non-covalently cross-linked supra-molecules formed by the self-assembly function of FTN-H. Upon the formation of the supra-molecule, its size is likely to be limited by the assembly properties of FTN-H, thereby keeping the self-assembled particles soluble. This study reports on the dual function of FTN-H for fusion expression and solubility enhancement of heterologous proteins: (i) high-affinity interaction with DnaK and (ii) formation of self-assembled supra-molecules with limited and constant sizes, thereby avoiding the undesirable formation of insoluble macro-aggregates of heterologous proteins.
引用
收藏
页码:3751 / 3762
页数:12
相关论文
共 38 条
  • [11] Bukau B., 1999, MOL CHAPERONES FOLDI, P3
  • [12] Binding of non-native protein to Hsp25 during heat shock creates a reservoir of folding intermediates for reactivation
    Ehrnsperger, M
    Graber, S
    Gaestel, M
    Buchner, J
    [J]. EMBO JOURNAL, 1997, 16 (02) : 221 - 229
  • [13] In vivo observation of polypeptide flux through the bacterial chaperonin system
    Ewalt, KL
    Hendrick, JP
    Houry, WA
    Hartl, FU
    [J]. CELL, 1997, 90 (03) : 491 - 500
  • [14] Protein quality control: Triage by chaperones and proteases
    Gottesman, S
    Wickner, S
    Maurizi, MR
    [J]. GENES & DEVELOPMENT, 1997, 11 (07) : 815 - 823
  • [15] Mutational analysis of the four α-helix bundle iron-loading channel of rat liver ferritin
    Guo, JH
    Juan, SH
    Aust, SD
    [J]. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1998, 352 (01) : 71 - 77
  • [16] HAASEPETTINGELL CA, 1988, J BIOL CHEM, V263, P4977
  • [17] Ferritins: Molecular properties, iron storage function and cellular regulation
    Harrison, PM
    Arosio, P
    [J]. BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 1996, 1275 (03): : 161 - 203
  • [18] Protein folding - Molecular chaperones in the cytosol: from nascent chain to folded protein
    Hartl, FU
    Hayer-Hartl, M
    [J]. SCIENCE, 2002, 295 (5561) : 1852 - 1858
  • [19] In vitro disulfide-coupled folding of guanylyl cyclase-activating peptide and its precursor protein
    Hidaka, Y
    Ohno, M
    Hemmasi, B
    Hill, O
    Forssmann, WG
    Shimonishi, Y
    [J]. BIOCHEMISTRY, 1998, 37 (23) : 8498 - 8507
  • [20] The molecular chaperone DnaJ is required for the degradation of a soluble abnormal protein in Escherichia coli
    Huang, HC
    Sherman, MY
    Kandror, O
    Goldberg, AL
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 2001, 276 (06) : 3920 - 3928