Nurmamet Amet1, Hsin-Fang Lee, Wei-Chiang Shen. 1. Department of Pharmacology and Pharmaceutical Sciences, University of Southern California School of Pharmacy, PSC 404B, 1985 Zonal Avenue, Los Angeles, California, 90089-9121, USA.
Abstract
PURPOSE: To demonstrate a high-level expression of transferrin (Tf)-based fusion proteins by inserting a helical linker between two protein domains. METHODS: Tf-based fusion proteins were designed to contain oligonucleotides encoding a helical linker inserted between the protein domains. Plasmid constructs were transfected into HEK293 cells and the secreted fusion proteins were purified from conditioned serum free media. Expression was assessed using both SDS-PAGE and Western Blot using anti-hGH, G-CSF, or Tf antibodies; protein bands were analyzed using Quantity One software. The function of fusion proteins consisting of human growth hormone (hGH) and Tf was evaluated in Nb2 cell proliferation assays. RESULTS: The fusion proteins containing a helical linker, hGH-(H4)(2)-Tf and Tf-(H4)(2)-hGH, were expressed 1.7-and 2.4-fold higher, respectively, with a twofold lower ED(50) than the hGH-Tf fusion protein without a helical linker. The Tf-(H4)(2)-G-CSF fusion protein exhibited a greater expression with an 11.2-fold increase compared with Tf-G-CSF fusion protein. CONCLUSIONS: The helical linker introduced in Tf-fusion proteins resulted in a high-level of expression with improved in vitro bioactivity. This approach provides a simple method to increase poor expression of other fusion proteins.
PURPOSE: To demonstrate a high-level expression of transferrin (Tf)-based fusion proteins by inserting a helical linker between two protein domains. METHODS: Tf-based fusion proteins were designed to contain oligonucleotides encoding a helical linker inserted between the protein domains. Plasmid constructs were transfected into HEK293 cells and the secreted fusion proteins were purified from conditioned serum free media. Expression was assessed using both SDS-PAGE and Western Blot using anti-hGH, G-CSF, or Tf antibodies; protein bands were analyzed using Quantity One software. The function of fusion proteins consisting of humangrowth hormone (hGH) and Tf was evaluated in Nb2 cell proliferation assays. RESULTS: The fusion proteins containing a helical linker, hGH-(H4)(2)-Tf and Tf-(H4)(2)-hGH, were expressed 1.7-and 2.4-fold higher, respectively, with a twofold lower ED(50) than the hGH-Tf fusion protein without a helical linker. The Tf-(H4)(2)-G-CSF fusion protein exhibited a greater expression with an 11.2-fold increase compared with Tf-G-CSF fusion protein. CONCLUSIONS: The helical linker introduced in Tf-fusion proteins resulted in a high-level of expression with improved in vitro bioactivity. This approach provides a simple method to increase poor expression of other fusion proteins.
Authors: M Ishikawa; A Nimura; R Horikawa; N Katsumata; O Arisaka; M Wada; M Honjo; T Tanaka Journal: J Clin Endocrinol Metab Date: 2000-11 Impact factor: 5.958
Authors: Igor A Kaltashov; Cedric E Bobst; Rinat R Abzalimov; Guanbo Wang; Burcu Baykal; Shunhai Wang Journal: Biotechnol Adv Date: 2011-05-17 Impact factor: 14.227