TY - JOUR
T1 - Active human ferritin H/L-hybrid and sequence effect on folding efficiency in Escherichia coli
AU - Lee, Jeewon
AU - Kim, Sung Woo
AU - Kim, Yang Hoon
AU - Ahn, Ji Young
N1 - Funding Information:
The work for stable synthesis of recombinant ferritin H/L-hybrid with bioactivity was supported by Bioprogen Co. The research for correlating H-chain sequence with intracellular solubility of various ferritin hybrids was supported by a Korea University grant.
PY - 2002
Y1 - 2002
N2 - 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 (295 mol 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.
AB - 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 (295 mol 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.
KW - Cytoplasmic solubility
KW - Ferritin H/L-hybrid
KW - Folding efficiency
KW - Intra-molecular interaction
KW - Iron storage capacity
UR - http://www.scopus.com/inward/record.url?scp=0036406209&partnerID=8YFLogxK
U2 - 10.1016/S0006-291X(02)02429-4
DO - 10.1016/S0006-291X(02)02429-4
M3 - Article
C2 - 12387819
AN - SCOPUS:0036406209
SN - 0006-291X
VL - 298
SP - 225
EP - 229
JO - Biochemical and biophysical research communications
JF - Biochemical and biophysical research communications
IS - 2
ER -