Transcript optimized expression enhancement for high-level production of proteins and protein domains

A method of preparing an expression vector, wherein the expression vector comprises, in order of position:

• a first nucleic acid sequence encoding a 5′ untranslated region of an expressed mRNA that comprises a ribosome binding site (RBS);
• a second nucleic acid sequence encoding a polypeptide tag;
• and a cloning site, wherein the cloning site enables a target protein coding sequence to be inserted into the vector in-frame with the second nucleic acid sequence to encode a fusion protein comprising the polypeptide tag and the target protein;
• and wherein the method comprises specifically modifying the nucleic acid sequence encoding (i) the 5′ untranslated region and (ii) the adjacent polypeptide tag to minimize RNA secondary structure both within and/or between these two regions of the mRNA.