Dual affinity fusion approach and its use to express recombinant human insulin-like growth factor II.
A dual affinity fusion concept has been developed in which the gene encoding the desired product is fused between two flanking heterologous genes encoding IgG- and albumin-binding domains. Using sequential IgG and serum albumin affinity chromatography, a full-length tripartite fusion protein is obtained. This approach was used to recover a full-length fusion product in Escherichia coli containing the human insulin-like growth factor II (IGF-II). Surprisingly, the recombinant IGF-II showed increased stability against proteolytic degradation in E. coli when produced as a dual affinity fusion protein, as compared to an N-terminal fusion protein. After site-specific cleavage of the tripartite fusion protein, IGF-II molecules with immunological and receptor binding activity were obtained without renaturation steps. The results demonstrate that proteins can fold into biologically active structures, even if provided with large flanking heterologous protein domains. The concept was further used to characterize the specific degradation of recombinant IGF-II in this heterologous host.
Hammarberg
B
B
Department of Biochemistry, Royal Institute of Technology, Stockholm, Sweden.
Nygren
P A
PA
Holmgren
E
E
Elmblad
A
A
Tally
M
M
Hellman
U
U
Moks
T
T
Uhlén
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010957:Plasmids; D011956:Receptors, Cell Surface / Q000378:metabolism; D017451:Receptors, Somatomedin; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D013002:Somatomedins / Q000235:genetics*
D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D011993:Recombinant Fusion Proteins; D013002:Somatomedins; D007335:Insulin-Like Growth Factor II