Insertion of the designed helical linker led to increased expression of tf-based fusion proteins.
OBJECTIVE
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.
Amet
Nurmamet
N
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California School of Pharmacy, PSC 404B, 1985 Zonal Avenue, Los Angeles, California, 90089-9121, USA.
Lee
Hsin-Fang
HF
Shen
Wei-Chiang
WC
D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D019382:Human Growth Hormone / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D011994:Recombinant Proteins; D014168:Transferrin / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology
D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014168:Transferrin; D019382:Human Growth Hormone; D016179:Granulocyte Colony-Stimulating Factor