Recombinant protein production in cultures of an Escherichia coli trp- strain.
Fermentation conditions were developed in order to achieve simultaneously a high biomass concentration and high-level expression of a hybrid cI-human insulin B peptide gene. In our system, this hybrid gene is under control of the Escherichia coli trp promoter, in a trp- derivative strain of E. coli W3110. The dual role of tryptophan concentration on cellular growth and hybrid gene regulation was studied in 10-1 batch fermentations. In the best batch conditions, a biomass concentration of 12 g dry weight/l can be obtained, and 0.53 g/l of cI-insulin B hybrid protein is produced. Tryptophan in the culture medium is consumed by the growing culture, until a level is reached that causes induction of the hybrid gene. Plasmid loss was detected, as only 62% of the cells retained the recombinant plasmid. In order to increase the hybrid protein production level, a fed-batch culture strategy was developed whereby the specific growth rate of the cells was restrained. Using the same amount of nutrients as in the batch fermentations, it was possible to increase the final biomass concentration to 20 g/l, plasmid-bearing cells in the population to 90% and recombinant hybrid protein to 1.21 g/l.
Gosset
G
G
Departamentos de Biología Molecular y Bioingeniería, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos.
de Anda
R
R
Cruz
N
N
Martínez
A
A
Quintero
R
R
Bolivar
F
F
D001709:Biotechnology; D003470:Culture Media; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D005285:Fermentation; D005947:Glucose; D006801:Humans; D007328:Insulin / Q000096:biosynthesis / Q000235:genetics; D007700:Kinetics; D010710:Phosphates; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics; D014364:Tryptophan / Q000378:metabolism; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D003470:Culture Media; D004268:DNA-Binding Proteins; D007328:Insulin; D010710:Phosphates; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C024788:ammonium phosphate; C016649:phage repressor proteins; D014364:Tryptophan; D005947:Glucose