The use of chimeric gene constructs to express a bacterial endoglucanase in mammalian cells.
The synthesis and secretion of a truncated Clostridium thermocellum endoglucanase (EGE') encoded by the celE' gene was investigated in Chinese hamster ovary (CHO) cells. Fusion genes consisting of the human growth hormone (hGH) gene and celE', transcribed from the SV40 early enhancer/promoter, were constructed and stably transfected into CHO cells. A gene consisting of celE' inserted into the first exon of the hGH gene resulted in the synthesis of truncated proteins (less than or equal to 22 kDa) lacking endoglucanase activity. Cloning celE' into the second exon of the hGH gene, resulted in the synthesis and secretion of a 50 kDa protein with endoglucanase activity. A 50 kDa protein was also synthesised by cells transfected with celE' cloned into the fifth exon of the hGH gene. However, despite a 5-fold increase in enzyme activity compared to the exon 2 transfected cell line less than 40% of the protein was secreted. Constructs devoid of introns, in which celE' was fused to the SV40 early promoter and to the rabbit beta-globin polyadenylation sequence resulted in a 2-18-fold increase in endoglucanase activity compared to the constructs containing introns. In addition more than 75% of the synthesised protein was secreted. Analyses of EGE' encoded mRNA from the transfected cell lines suggests that the presence of introns results in the aberrant splicing of message by the use of cryptic splice sites in the celE' gene. These results demonstrate that introns are not required for the efficient expression of a bacterial endoglucanase in mammalian cells, rather introns appear to reduce expression of the encoded protein.
Hall
J
J
Department of Biological & Nutritional Sciences, University of Newcastle upon Tyne, UK.
Hirst
B H
BH
Hazlewood
G P
GP
Gilbert
H J
HJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D016466:CHO Cells; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D006224:Cricetinae; D005786:Gene Expression Regulation / Q000235:genetics*; D013006:Growth Hormone / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007438:Introns / Q000235:genetics*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics
D011993:Recombinant Fusion Proteins; D013006:Growth Hormone; D002480:Cellulase