A Bacillus subtilis fusion protein system to produce soybean Bowman-Birk protease inhibitor.
A fusion protein based expression system was developed in the Gram-positive bacterium Bacillus subtilis to produce the soybean Bowman-Birk protease inhibitor (sBBI). The N-terminus of the mature sBBI was fused to the C-terminus of the 1st cellulose binding domain linker (CBD linker) of the BCE103 cellulase (from an alkalophilic Bacillus sp.). The strong aprE promoter was used to drive the transcription of the fusion gene and the AprE signal sequence was fused to the mature BCE103 cellulase for efficient secretion of the fusion protein into the culture medium. It was necessary to use a B. subtilis strain deficient in nine protease genes in order to reduce the proteolytic degradation of the fusion protein during growth. The fusion protein was produced in shake flasks at concentrations >1g/L. After growth, the sBBI was activated by treatment with 2-mercaptoethanol to allow the disulfide bonds to form correctly. An economical and scalable purification process was developed to purify sBBI based on acid precipitation of the fusion protein followed by acid/heat cleavage of the fusion protein at labile Asp-Pro bonds in the CBD linker. If necessary, non-native amino acids at the N- and C-termini were trimmed off using glutamyl endopeptidase I. After purification, an average of 72 mg of active sBBI were obtained from 1L of culture broth representing an overall yield of 21% based on the amount of sBBI activated before purification.
Vogtentanz
Gudrun
G
Genencor, Danisco USA, Inc., 925 Page Mill Road, Palo Alto, CA 94304, USA.
Collier
Katherine D
KD
Bodo
Michael
M
Chang
Judy H
JH
Day
Anthony G
AG
Estell
David A
DA
Falcon
Brandy C
BC
Ganshaw
Grant
G
Jarnagin
Alisha S
AS
Kellis
James T
JT
Kolkman
Marc A B
MA
Lai
Cindy S
CS
Meneses
Renato
R
Miller
Jeffrey V
JV
de Nobel
Hans
H
Power
Scott
S
Weyler
Walter
W
Wong
David L
DL
Schmidt
Brian F
BF
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D002480:Cellulase / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000737:chemistry; D013381:Subtilisins / Q000235:genetics; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D002480:Cellulase; D012697:Serine Endopeptidases; D013381:Subtilisins; C028344:glutamyl endopeptidase