Vector design for optimal protein expression.
Many DNA constructs are generated for protein expression studies. Translational properties and mRNA stability are crucial aspects that have to be accounted for during DNA construction. An optimized vector for protein overexpression studies is described considering elements in the mature mRNA that influence translatability and stability. Recommendations regarding vector construction for Xenopus laevis embryo injection are provided, based on literature and experimental data. The 5'untranslated region (5'UTR) should be non-regulated, short, unstructured, and without AUG codons. The sequence around the start codon should match the initiation context of the species studied (ACCAUGG, for vertebrates), and the open reading frame should be cloned with its own stop codon, followed by a G or A residue. Furthermore, the 3'UTR should be non-regulated, and a strong polyadenylation signal must be included in DNA vectors. In RNA template vectors, the presence of a poly(A) or AC tail is essential for stability, as well as for translation efficiency in mRNA injection experiments. These aspects result in high-level expression of exactly the desired protein. Easily obtainable examples of the sequences [5'UTR, 3'UTR, and poly(A) signal] are suggested.
van der Velden
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AW
Utrecht University, The Netherlands.
Voorma
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HO
Thomas
A A
AA
D020413:3' Untranslated Regions / Q000235:genetics; D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular; D003062:Codon; D004262:DNA Restriction Enzymes; D004355:Drug Stability; D004625:Embryo, Nonmammalian / Q000378:metabolism; D039562:Eukaryotic Initiation Factor-4F; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D016366:Open Reading Frames; D010448:Peptide Initiation Factors; D011061:Poly A / Q000378:metabolism; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D012315:RNA Caps / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes; D014162:Transfection; D014982:Xenopus laevis / Q000196:embryology
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D003062:Codon; D039562:Eukaryotic Initiation Factor-4F; D008164:Luminescent Proteins; D010448:Peptide Initiation Factors; D011506:Proteins; D012315:RNA Caps; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D011061:Poly A; D004262:DNA Restriction Enzymes