10.6084/m9.figshare.5124421.v1 Ying Q. Ying Q. Zhang C. Zhang C. Guo F. Guo F. Wang S. Wang S. Bie X. Bie X. Lu F. Lu F. Lu Z. Lu Z. Supplementary Material for: Secreted Expression of a Hyperthermophilic α-Amylase Gene from <b><i>Thermococcus</i></b> sp. HJ21 in <b><i>Bacillus subtilis</i></b> Karger Publishers 2013 Hyperthermophilic α-amylase Bacillus subtilis Promoter Signal peptide Secreted expression 2013-03-08 00:00:00 Dataset https://karger.figshare.com/articles/dataset/Supplementary_Material_for_Secreted_Expression_of_a_Hyperthermophilic_-Amylase_Gene_from_b_i_Thermococcus_i_b_sp_HJ21_in_b_i_Bacillus_subtilis_i_b_/5124421 The hyperthermophilic α-amylase from <i>Thermococcus</i> sp. HJ21 possesses unique traits (Ca<sup>2+</sup>-independent thermostability and optimal temperature of 95°C) that make it a great potential candidate for use in the food industry. However, this Archaea isolated from a deep-sea thermal vent requires strict control of culture conditions and produces only small amounts of α-amylase. To solve these problems, the α-amylase gene was cloned and expressed in <i>Bacillus subtilis</i>, which is an ideal food-grade host for heterologous protein expression. To express high levels of this α-amylase in <i>B. subtilis</i>, the promoters P<sub><i>grac</i></sub>, P<sub><i>xylA</i></sub>, P43, and P<sub><i>hag</i></sub> were used to construct four different expression vectors for testing. The vector containing the P<sub><i>xylA</i></sub> promoter was found to have the highest transcriptional activity and produce the highest amylase activity (19.6 U/ml). To test the secretion efficiency of signal peptides in <i>B. subtilis</i>, three signal peptides were cloned and fused to the α-amylase gene (lacking its native signal peptide). The optimal signal peptide was S<sub><i>amyQ</i></sub>, with a secretion efficiency of approximately 90%. These results indicate that the promoter P<sub><i>xylA</i></sub> and signal peptide S<sub><i>amyQ</i></sub> tested in this study may be useful for the expression and secretion of archaeal proteins in <i>B. subtilis</i>.