Effect of substrate feed rate on recombinant protein secretion, degradation and inclusion body formation in Escherichia coli.
The effect of changes in substrate feed rate during fedbatch cultivation was investigated with respect to soluble protein formation and transport of product to the periplasm in Escherichia coli. Production was transcribed from the P(malK) promoter; and the cytoplasmic part of the production was compared with production from the P(lacUV5) promoter. The fusion protein product, Zb-MalE, was at all times accumulated in the soluble protein fraction except during high-feed-rate production in the cytoplasm. This was due to a substantial degree of proteolysis in all production systems, as shown by the degradation pattern of the product. The product was also further subjected to inclusion body formation. Production in the periplasm resulted in accumulation of the full-length protein; and this production system led to a cellular physiology where the stringent response could be avoided. Furthermore, the secretion could be used to abort the diauxic growth phase resulting from use of the P(malK) promoter. At high feed rate, the accumulation of acetic acid, due to overflow metabolism, could furthermore be completely avoided.
Boström
Maria
M
The Swedish Centre for Bioprocess Technology, KTH, AlbaNova University Center, Stockholm, 106-91, Sweden.
Markland
Katrin
K
Sandén
Anna Maria
AM
Hedhammar
My
M
Hober
Sophia
S
Larsson
Gen
G
D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D046508:Culture Techniques; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D002479:Inclusion Bodies / Q000378:metabolism*; D007700:Kinetics; D030781:Organisms, Genetically Modified; D033902:Periplasmic Binding Proteins / Q000378:metabolism; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013997:Time Factors; D014158:Transcription, Genetic
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; C068021:MalK protein, Bacteria; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; D005947:Glucose