Heterologous gene expression using self-assembled supra-molecules with high affinity for HSP70 chaperone.
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.
Ahn
Ji-Young
JY
Department of Chemical and Biological Engineering, Korea University Anam-Dong 5-1, Sungbuk-Ku, Seoul 136-713, South Korea.
Choi
Hyung
H
Kim
Yang-Hoon
YH
Han
Kyung-Yeon
KY
Park
Jin-Seung
JS
Han
Sung-Sik
SS
Lee
Jeewon
J
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001665:Binding Sites; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005293:Ferritins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000648:ultrastructure; D016415:Sequence Alignment
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D005293:Ferritins; C063842:dnaK protein, E coli