Effect of upstream sequence modifications in pET vectors on the yield of recombinant proteins

Increasing the yield of recombinant proteins can save time and materials and, consequently, any strategy that seems promising for achieving this objective is worth investigating. Lately, it has been proposed that modifications of upstream sequences from pET vectors would enhance the transcription efficiency. To validate this approach, we expressed a pool of constructs in modified pET vectors and compared the yields with those obtained using unmodified pET-derived vectors. Constructs were selected according to their different structural properties and protein yields. In our experimental setting, the results were generally deceiving. However, improvements were observed when additional elements of the construct were optimized concurrently. In particular, repositioning the His-tag from the C-terminus to the N-terminus positively affected the final protein yield. Taken together, these findings indicate that the modified vectors developed in this study represent one of several factors to consider during the initial screening aimed at identifying optimal expression conditions for a new construct, rather than a broadly applicable solution for enhancing the yield of any protein construct.