Active human ferritin H/L-hybrid and sequence effect on folding efficiency in Escherichia coli.
In Escherichia coli, the recombinant human L-chain ferritin was synthesized in the form of inclusion bodies under the control of T7 promoter system. We developed a recombinant ferritin H/L-hybrid by a direct gene fusion between H- and L-chain subunits. Surprisingly, the presence of heavy-chain polypeptide at the amino terminus of light chain significantly increased the cytoplasmic solubility of the recombinant ferritin hybrid, i.e., more than 80% of synthesized ferritin hybrid was soluble in intracellular region regardless of the changes in cell growth and gene expression conditions such as type of inducer, growth media, culture scale, etc. The soluble ferritin H/L-hybrid was biologically active with the iron storage capacity (295mol Fe(+3) per mol H/L-hybrid) equivalent to ferritin standard. Different types of hybrid mutants were also developed using various H-chain derivatives. Comparison of the intracellular solubilities of the synthesized hybrid mutants showed that the N-terminus four helices of heavy subunit were of crucial importance in maintaining the high solubility in E. coli cytoplasm. Consequently, the increased solubility of the ferritin hybrid seems to be related to such H-chain sequence that forms ferroxidase center and promotes effective intra-molecular interaction with L-chain domain of H/L-hybrid for enhancing the folding efficiency.
Lee
Jeewon
J
Department of Chemical and Biological Engineering, Korea University, 1, 5-Ka, Anam-Dong, Sungbuk-Ku, Seoul 136-701, Republic of Korea. leejw@korea.ac.kr
Kim
Sung Woo
SW
Kim
Yang Hoon
YH
Ahn
Ji Young
JY
D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D004926:Escherichia coli / Q000235:genetics*; D005293:Ferritins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility
D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D005293:Ferritins; D007501:Iron