Recombinant human insulin IX. Investigation of factors, influencing the folding of fusion protein-S-sulfonates, biotechnological precursors of human insulin.
The peculiarities of molecular structures and the influence of reaction conditions on the folding efficiency of fusion proteins-biotechnological precursors of human insulin, expressed in Escherichia coli as inclusion bodies have been investigated. The fusion proteins contained proinsulin sequence with various leader peptides connected by an Arg residue to the insulin B-chain. The kind and the size of leader peptide do not have essential influence on folding efficiency. However, the efficiency of protein folding depends on the location of the (His)6 site, which is used for metal-chelating affinity chromatography. In our study the protein folding depends on the reaction medium composition (including additives), the presence of accompanied cell components, pH, temperature, concentrations of protein, and redox agents. A negative influence of nucleic acid and heavy metal ions on folding has been found. S-sulfonated fusion protein has proinsulin-like secondary structure (by CD-spectroscopy data) that is the key point for 95% efficient folding proceeding. Folded fusion proteins are transformed into insulin by enzymatic cleavage.
Tikhonov
Roman V
RV
M.M Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Street 16/10, GSP, V-437, 117997, Moscow, Russia.
Pechenov
Sergey E
SE
Belacheu
Irina A
IA
Yakimov
Sergey A
SA
Klyushnichenko
Vadim E
VE
Tunes
Heloisa
H
Thiemann
Josef E
JE
Vilela
Luciano
L
Wulfson
Andrey N
AN
D000595:Amino Acid Sequence; D001709:Biotechnology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006801:Humans; D007328:Insulin / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013451:Sulfonic Acids / Q000378:metabolism*
D007328:Insulin; D011498:Protein Precursors; D011994:Recombinant Proteins; D013451:Sulfonic Acids