title: Transcriptional regulation of gene expression by the coding sequence: An attempt to enhance expression of human AChE.
abstract: In a previous report, Morel and Massoulié showed that Bungarus AChE (bBAChE) is produced more efficiently than rat AChE in various expression systems, mainly because the Bungarus coding sequence exerts a stimulatory effect on transcription (Morel and Massoulié, 2000). They reported that a 5' Bungarus fragment could partially transfer this property to a CAT expression vector. This appeared to offer the possibility of increasing the production of recombinant proteins. In the present paper, we show that insertion of this fragment in the transcribed region, before the polyadenylation site, may have either stimulatory or inhibitory effects, depending on the vector and on the reporter gene. Since the stimulatory effect of Bungarus coding region could not be attached to a small number of discrete motifs, we reasoned that it might result from a general feature of the sequence. Therefore it might be possible to partially transfer this property to the very homologous human AChE (hHAChE) coding sequence by modifications based on synonymous codons, which increased nucleotide identity between the 5' fragment (721 nucleotides) of bBAChE and hHAChE from 71% to 85%. The production of human AChE in transfected COS cells was increased nearly 2-fold with this modified construct, but still remained about 4-fold smaller than that of Bungarus AChE. There was no change in expression level in transformed Pichia pastoris. We thus confirm that coding sequences can strongly influence gene expression, but in a manner that depends on the context and cannot yet be predicted.
mesh_terms: D000110:Acetylcholinesterase / Q000096:biosynthesis* / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D017838:Bungarus / Q000235:genetics / Q000378:metabolism; D019556:COS Cells / Q000201:enzymology; D004795:Enzyme Stability; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000235:genetics; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames / Q000235:genetics*; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics*; D012016:Reference Values; D017385:Sequence Homology; D015533:Transcriptional Activation
chemical_list: D011994:Recombinant Proteins; D000110:Acetylcholinesterase