Effects of signal peptide changes on the secretion of bovine somatotropin (bST) from Escherichia coli |
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Authors: | Klein Barbara K; Polazzi Joseph O; Devine Catherine S; Rangwala Shaukat H; Olins Peter O |
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Affiliation: | Monsanto Corporate Research, Monsanto Company 700 Chesterfield Village Parkway, St Louis, MO 63198. USA |
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Abstract: | Bovine somatotropin (bST) was secreted from Escherichia coliat moderate levels of 12 µg/ml/OD using expressionvectors in which the bST gene was fused to the lamB secretionsignal. To study the secretion properties of bST in E.coli further,two approaches for modifying the secretion signal were employed.In the first case, fusion proteins were constructed with sixalternative bacterial secretion signals: three from E.coli proteins(HisJ, MalE and OmpA), two from bacteriophage proteins (M13coat protein and PA-2 Lc) and one from the chitinase A proteinof Serratia marcescens. The results, as monitored by Westernblot analysis of both total cell protein and the periplasmicfraction, showed that these changes in the secretion signaldid not significantly affect the secretion properties of bST.In the second approach, a library of random mutations was createdin the lamB secretion signal and 200 independent clones werescreened. The level of secreted bST was determined by growingindividual clones in duplicate in microtiter wells, inducingprotein expression and measuring the bST released by osmoticshock using a particle concentration fluorescent immunoassay.The secretion properties of several novel variants in the LamBsignal peptide are presented. |
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Keywords: | bST/ E coli/ secretion/ signal peptide |
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