Human growth hormone expressed in tobacco cells as an arabinogalactan-protein fusion glycoprotein has a prolonged serum life.
Therapeutic proteins with molecular weights lower than 40 kDa often have short serum half-lives due to their susceptibility to serum proteases and rapid renal clearance. Chemical derivatization, such as PEGylation, or expression as serum albumin fusions increases molecular mass and overcome these problems but at the expense of decreased bioactivity. Here we applied a new method that yields biologically potent recombinant human growth hormone (rhGH) with increased serum half-life when expressed as an arabinogalactan-protein (AGP) in tobacco BY-2 cells. Thus, rhGH was expressed with 10 repeats of the AGP glycomodule Ser-Hyp (SO) at the C-terminus (rhGH-(SO)(10)). We also expressed rhGH as an AGP-enhanced green fluorescent protein (EGFP) fusion, designated rhGH-(SO)(10)-EGFP, to assess the cellular distribution of the glycoprotein, which was mainly extracellular. Recombinant hGH-(SO)(10) bound the hGH receptor with an affinity similar to that of a rhGH standard, stimulated the same intracellular signaling pathway as hGH, but possessed an in vivo serum half-life more than sixfold that of the hGH control. Furthermore, rhGH-(SO)(10) gave a 500 fold greater secreted yield than the non-glycosylated control rhGH that was also targeted for secretion. Detailed analysis of the rhGH-(SO)(10) glycans indicated a conserved structure with relatively little microheterogeneity and an average size of 25 monosaccharide residues. These results were consistent with earlier work expressing interferon alpha 2b as an AGP chimera and further demonstrate the feasibility of this approach to the production of long-acting, biologically potent therapeutic proteins by plant cells.
Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA. jxu@astate.edu
Jianfeng
J
Xu
Shigeru
S
Okada
Li
L
Tan
Kenneth J
KJ
Goodrum
John J
JJ
Kopchick
Marcia J
MJ
Kieliszewski
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000737:chemistry; D005685:Galactans / Q000737:chemistry*; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006207:Half-Life; D019382:Human Growth Hormone / Q000097:blood / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000493:pharmacokinetics; D006801:Humans; D007739:L Cells; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification / Q000493:pharmacokinetics*; D015398:Signal Transduction / Q000187:drug effects; D014026:Nicotiana / Q000166:cytology
D017077:Culture Media, Conditioned; D005685:Galactans; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D019382:Human Growth Hormone; D049452:Green Fluorescent Proteins; C005653:arabinogalactan