Efficient secretory production of alkaline phosphatase by high cell density culture of recombinant Escherichia coli using the Bacillus sp. endoxylanase signal sequence.
New secretion vectors containing the Bacillus sp. endoxylanase signal sequence were constructed for the secretory production of recombinant proteins in Escherichia coli. The E. coli alkaline phosphatase structural gene fused to the endoxylanase signal sequence was expressed from the trc promoter in various E. coli strains by induction with IPTG. Among those tested, E. coli HB101 showed the highest efficiency of secretion (up to 25.3% of total proteins). When cells were induced with 1 mM IPTG, most of the secreted alkaline phosphatase formed inclusion bodies in the periplasm. However, alkaline phosphatase could be produced as a soluble form without reduction of expression level by inducing with less (0.01 mM) IPTG, and greater than 90% of alkaline phosphatase could be recovered from the periplasm by the simple osmotic shock method. Fed-batch cultures were carried out to examine the possibility of secretory protein production at high cell density. Up to 5.2 g/l soluble alkaline phosphatase could be produced in the periplasm by the pH-stat fed-batch cultivation of E. coli HB101 harboring pTrcS1PhoA. These results demonstrate the possibility of efficient secretory production of recombinant proteins in E. coli by high cell density cultivation.
Department of Chemical Engineering and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, Taejon.
J H
JH
Choi
K J
KJ
Jeong
S C
SC
Kim
S Y
SY
Lee
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D043364:Endo-1,4-beta Xylanases; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014995:Xylosidases / Q000235:genetics*
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases