کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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6452034 | 1416985 | 2017 | 7 صفحه PDF | دانلود رایگان |
- Split GFP assay can be used to monitor and quantify secreted proteins and enzymes.
- Activity and amount of protein in culture supernatants correlate with fluorescence signals.
- The assay is applicable for high throughput screening of large libraries.
The large-scale industrial production of proteins requires efficient secretion, as provided, for instance, by the Sec system of Gram-positive bacteria. Protein engineering approaches to optimize secretion often involve the screening of large libraries, e.g. comprising a target protein fused to many different signal peptides. Respective high-throughput screening methods are usually based on photometric or fluorimetric assays enabling fast and simple determination of enzymatic activities. Here, we report on an alternative method for quantification of secreted proteins based on the split GFP assay. We analyzed the secretion by Bacillus subtilis of a homologous lipase and a heterologous cutinase by determination of GFP fluorescence and enzyme activity assays. Furthermore, we identified from a signal peptide library a variant of the biotechnologically relevant B. subtilis protein swollenin EXLX1 with up to 5-fold increased secretion. Our results demonstrate that the split GFP assay can be used to monitor secretion of enzymatic and non-enzymatic proteins in B. subtilis in a high-throughput manner.
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Journal: Journal of Biotechnology - Volume 258, 20 September 2017, Pages 110-116