Highly efficient expression of functional recombinant human keratinocyte growth factor 1 and its protective effects on hepatocytes.
Three forms of recombinant human keratinocyte growth factor 1 (rhKGF1) with or without the native signal peptide or a 23-amino acid truncation were expressed in Spodoptera frugiperda 9 (Sf9) cells by designing with insect codon usage. Immunoblotting demonstrated that these rhKGF1 proteins were recognized by a human anti-KGF1 antibody. The multiplicity of infection and timing of harvest had a significant effect on protein yield, protein quality, and cytotoxicity. Our results indicated that the native signal peptide directed KGF1 secretion from insect cells, reaching a maximum at 60 h postinfection. Although secretion of rhKGF1194 was less efficient than that of rhKGF1163 and rhKGF1140, protein secretion is an attractive pathway for simple purification of biologically active rhKGF1 at a high yield. Moreover, the sizes of rhKGF1194 and rhKGF1163 were similar (20 kDa), suggesting that the signal peptide may be recognized and removed in Sf9 cells. A 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay was used to analyze the biological function of rhKGF1, indicating that the three forms of rhKGF1 had a similar mitogenic function in BaF3 cells. Furthermore, to elucidate the effect of rhKGF1 on cytoprotection of liver cells, we used KGF1 pretreatment of an acute liver injury model. The results indicated that rhKGF1 prevented necrosis and apoptosis of CCl4-treated HL7702 cells in vitro and in vivo. These results suggest that KGF1 may be a candidate therapeutic drug for acute liver injury.
School of Pharmaceutical Science, Key Laboratory of Biotechnology and Pharmaceutical Engineering of Zhejiang Province, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, China.
Ping
P
Xue
Xiaojing
X
Zhu
Junqing
J
Shi
Hongqi
H
Fu
Jian
J
Zhang
Min
M
Liu
Chao
C
Jiang
Xiaokun
X
Li
D000818:Animals; D002460:Cell Line; D019610:Cytoprotection; D051523:Fibroblast Growth Factor 7 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D022781:Hepatocytes / Q000187:drug effects* / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D018411:Spodoptera
C496453:FGF7 protein, human; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D051523:Fibroblast Growth Factor 7