Expression and secretion of an alpha-amylase gene from a native strain of Bacillus licheniformis in Escherichia coli by T7 promoter and putative signal peptide of the gene.
The gene encoding a hyperthermostable alpha-amylase from a Bacillus licheniformis native strain was cloned in pET24d transcription vector containing T7 promoter, and expressed in Escherichia coli BL21(DE3) cells. Having confirmed the alpha-amylase activity through activity staining method on SDS-PAGE gel, the yields of production were determined in two separated intra and inter-cellular phases and compared using enzymatic assay methods. Extracellular production of the active recombinant enzyme implies the recognition of the putative signal peptide of this Bacillus sp. by E. coli secretory system. This may be because of the amino acid sequence of this signal peptide which covers all the structural parameters of a standard signal peptide processed by Lep B, the major signal peptidase in E. coli secretory system. This study recommends the use of this signal peptide for extracellular production of other foreign proteins in E. coli.
Shahhoseini
M
M
Institute of Biochemistry and Biophysics, Faculty of Sciences, University of Tehran, Tehran, Islamic Republic of Iran. hoseini@ibb.ut.ac.ir
Ziaee
A-A
AA
Ghaemi
N
N
D000595:Amino Acid Sequence; D001407:Bacillus / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007785:Lactose / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D000516:alpha-Amylases; D007785:Lactose