Vector design for optimal protein expression

Many DNA constructs are generated for protein expression studies. Translati onal properties and mRNA stability are crucial aspects that have to be acco unted for during DNA construction. An optimized vector for protein over exp ression studies is described considering elements in the mature mRNA that i nfluence translatability and stability. Recommendations regarding vector co nstruction for Xenopus laevis embryo injection are provided, based on liter ature and experimental data. The 5 ' untranslated region (5 ' UTR) should h e 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 mu st be included in DNA vectors, In RNA template vectors, the presence of a p oly(A) or AC tail is essential for stability. as well as for translation ef ficiency in mRNA injection experiments. These aspects result in high-level expression of exactly the desired protein. Easily obtainable examples of th e sequences [5 ' UTP, 3 ' UTR, and poly(A) signal] are suggested.