The N-terminal signal sequence and the last 98 amino acids are not essential for the secretion of Bacillus sp. TS-23 alpha-amylase in Escherichia coli.
A truncated Bacillus sp. TS-23 alpha-amylase gene lacking 96 and 294 bp at its 5' and 3' end respectively was prepared by polymerase chain reaction and cloned into Escherichia coli expression vector, pQE-30, under the control of T5 promoter. SDS-PAGE and activity staining analyses showed that the His6-tagged amylase had a molecular mass of approximately 54 kDa. Isopropyl-beta-d-thiogalactopyranoside (IPTG) induction of E. coli M15 cells bearing the recombinant plasmid resulted in the extracellular production of active amylase. Western blot analysis also revealed that the truncated amylase was present in the periplasmic space and culture medium.
Lo
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HF
Department of Food and Nutrition, Hung-Kuang Institute of Technology, Shalu, Taichung 433, Taiwan.
Lin
L L
LL
Li
C C
CC
Hsu
W H
WH
Chang
C T
CT
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005822:Genetic Vectors; D008970:Molecular Weight; D010957:Plasmids; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D014169:Transformation, Bacterial; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000516:alpha-Amylases