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> Ying Q. Zhang C. Guo F. Wang S. Bie X. Lu F. Lu Z. 10.6084/m9.figshare.5124421.v1 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>. 2013-03-08 00:00:00 Hyperthermophilic α-amylase Bacillus subtilis Promoter Signal peptide Secreted expression