Several protein expression systems can be used to produce recombinant proteins in required quantitites for studying their functions and properties for biotechnological applications. In view of the widespread use of His-tagged expressions, to understand the effect of His-tags on the protein structure and function is essential. Although, they are often used to facilitate expression and purification of chimeric proteins, the location of His tail has the potential to interfere with the function and structure of the related protein. Therefore, the decision regarding the relative positioning of the polyhistine tags remains difficult and depends on the primary structure and conformation of the protein. Here, we reported the effect of hexahistidine-tag position on the catalytic activity and solubility of formate dehydrogenase from Burkholderia dolosa PC543, as compared with other reported enzymes. To do this, the fdh gene was subcloned into the pET14b and pET22b (+) vectors to be able to express the protein with N- or C-terminal His tagged form in E. coli BL21(DE3), respectively. Then, both FDHs were purified and analyzed. Our results indicated that the C-terminal His tagged enzyme displayed higher activity and solubility compared with the N-terminal His tagged variant obtained as an inclusion body. We conclude that the previously reported enzymes and also FDHs could be affected by his tag position in several manners due to the enzyme origin. This study provides a deeper understanding about his tail effect on terminal fragments of bacterial FDHs and helps to engineer them.
Anahtar Kelimeler: N-/C- terminal his tag, Formate dehydrogenase, Solubility and Activity, Burkholderia dolosa PC543, Recombinant protein production
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