Dual Affinity Fusion Approach and Its Use to Express Recombinant Human Insulin-Like Growth Factor II

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 86; no. 12; pp. 4367 - 4371
• Main Authors: Hammarberg, Bjorn, Nygren, Per-Ake, Holmgren, Erik, Elmblad, Anette, Tally, Michael, Hellman, Ulf, Moks, Tomas, Uhlen, Mathias
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.06.1989; National Acad Sciences
• Subjects: Amino Acid Sequence; Base Sequence; Biochemistry; Biological and medical sciences; Chromatography; Disulfides; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene expression; gene fusion; Genes; Genetic Vectors; Health savings accounts; Humans; immunoglobulin G; Insulin-Like Growth Factor II - biosynthesis; Insulin-Like Growth Factor II - genetics; Insulin-Like Growth Factor II - isolation & purification; insulin-like growth factor-II; Material degradation; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Molecules; Plasmids; Promoter regions; Proteins; Receptors; Receptors, Cell Surface - metabolism; Receptors, Somatomedin; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - isolation & purification; Restriction Mapping; serum albumin; Somatomedins - genetics; Escherichia coli; Albumin; IgG; Gene organization; Method; Flanking sequence; Gene expression; Insulin like growth factor 2; Immunological method; Mutagenesis; Gel filtration; Bacteria; Escherichieae; Vector; Enterobacteriaceae
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.86.12.4367

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.