Production of recombinant human epidermal growth factor using Ssp dnaB mini-intein system.
Chemical-enzymatic synthesis of human Epidermal Growth Factor (hEGF) cDNA has been performed, following by cloning into expression vector pTWIN1 (New England Biolabs). The resulting recombinant fusion protein expressed in Escherichia coli consisted of the N-terminal chitin-binding domain, mini-intein Ssp dnaB domain and hEGF polypeptide at the C-terminus. In this construct, mini-intein Ssp dnaB played a role of catalytically active subunit capable under certain conditions of autocatalytic cleavage resulting in separation of the target protein. As the hybrid protein had several cysteins in its sequence-one in chitin-binding domain, one in mini-intein and six in hEGF, it was necessary to work out optimal scheme for refolding and purification of the recombinant hEGF. As a result of this work, two schemes of the recombinant hEGF purification have been developed: according to the first scheme, the recombinant protein with reduced cysteins is bound to the chitin column, the hEGF is cleaved off and eluted, and then refolded to form appropriate cystein bridges. In the second scheme, the entire hybrid protein is first refolded to form disulfide bonds and then loaded to affinity resin; the recombinant hEGF is cleaved off and eluted in its native state. In spite of the fact that the first scheme is more common and suitable for a variety of recombinant proteins, in case of recombinant hEGF, the second scheme proved to be more productive and cost-effective.
Esipov
Roman S
RS
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow 117997, Russia. esipov@ibch.ru
Stepanenko
Vasily N
VN
Chupova
Larisa A
LA
Boyarskikh
Uljana A
UA
Filipenko
Maxim L
ML
Miroshnikov
Anatoly I
AI
D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary; D053488:DnaB Helicases / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D006801:Humans; D047668:Inteins; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; C506730:dnaB protein, E coli; D053488:DnaB Helicases