Although it is known that expression at lower temperatures may increase the proportion of soluble protein [12] an optimization of the expression parameters was not attempted in this study as this greatly depends on the expressed protein.
In addition to presenting an optimized protocol for expression and purification of PglB, these results present a general guide for the systematic optimization of the expression, purification, and stability of a large, transmembrane protein.
Yields of protein were low (0.1 2 mg/l), however, it is likely that this could be improved upon by codon optimization of the expression constructs for E. coli if higher yields are required [53], [54].
The data from the optimization of the expression system shows that the secreted NAs (constructs 2 and 4) are highly active and soluble as the activity in the media was not decreased by ultracentrifugation, in contrast to membrane bound NA expressed in insects cells[16].
Lastly, continued optimization of the expression cassette design might be predicted to further improve the yield of FIX protein per transduced cell.
Altogether, we are confident that the present vector set combined with the L. lactis expression host has the potential to become a very useful tool in optimization of the expression, purification and functional analysis of extracytoplasmic bacterial proteins.
Our experimental results show that signal peptide optimization in the expression plasmid, addition of the gene propeptide, and proper gene dosage significantly increased RML expression level and enhanced the enzymatic properties.
In order to directly assess the effects of coding sequence optimization on the expression of the gene and the accumulation of its product, we compared the performance of the optimized gene construct (oACHE-S) to its native counterpart (hACHE-S) in N. benthamiana by transient expression assays (Fig. 3B) as well as in stable transformants (Fig. 3C).
First, the effect of the codon-optimization on the expression of the enzyme was evaluated.
