Heterologous expression of proteins often pursues high expression levels, but it can easily result in misfolding and loss of biological function. L-α-glycerophosphate oxidase (GlpO) is a flavin adenine dinucleotide (FAD)-dependent oxidase which is widely used in the clinical determination of triglycerides. We found that the total enzymatic activity of GlpO expressed in Escherichia coli (E. coli) was extremely low, probably due to the absence of FAD cofactors and the misfolding of GlpO at a high synthesis rate. Therefore, decreasing the expression rate was used to improve the activity of GlpO. The specific activity of GlpO expressed on the pUC19 vector with lac promotor was approximately 30 times higher than that expressed on the pET28a vector with T7 promotor, but the expression levels of GlpO on the two vectors were completely opposite. It indicated that the specific activity of GlpO was increased as the expression level decreased. However, too low expression greatly influences the total amount and activity of the functional enzyme. In order to resolve this problem, two new plasmids, GlpO-CG4 and GlpO-CG6, were constructed by inserting 4 or 6 nucleotides, respectively, between the ribosome binding site (RBS) and the start code (ATG) on pET28a. Compared with the expression on the GlpO-pET vector, the expression rates of GlpO on the GlpO-CG4 and GlpO-CG6 were dramatically decreased. The total activity of GlpO expressed on GlpO-CG6 was 11 times and 1.5 times higher than that expressed on the GlpO-pET and GlpO-pUC, respectively. Results suggest that the activity of GlpO can be improved by decreasing the expression rate.
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