Temperature-induced production of recombinant human insulin in high-cell density cultures of recombinant Escherichia coli.
The construction of expression vectors encoding either the human insulin A- or B-chains fused to a synthetic peptide and the temperature-induced expression of the recombinant genes in Escherichia coli are reported. Using this two-chain approach we also describe the separate isolation of the insulin A- and B-chains from inclusion bodies and their subsequent assembly into native human insulin. The production of the insulin fusion proteins were carried out in high-cell density fed-batch cultures using a synthetic medium with glucose as sole carbon and energy source. The expression of the recombinant genes by temperature-shift in high-cell density cultures of recombinant E. coli resulted in product yields of grams per litre of culture broth, e.g. 4.5 g of insulin B-chain fusion protein per litre of culture broth. This translates into an expression yield of about 800 mg of the insulin B-chain per litre of culture. Under similar cultivation conditions the expression yield of the insulin A-chain corresponds to approximately 600 mg per litre of culture. The metabolic burden imposed on the recombinant cells during temperature-induced production of insulin fusion proteins in high-cell density cultures is reflected in an increased respiratory activity and a reduction of the biomass yield coefficient with respect to glucose.
GBF National Research Center for Biotechnology, Biochemical Engineering Division, Braunschweig, Germany.
M
M
Schmidt
K R
KR
Babu
N
N
Khanna
S
S
Marten
U
U
Rinas
D000595:Amino Acid Sequence; D019149:Bioreactors; D001709:Biotechnology; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013696:Temperature
D003470:Culture Media; D007328:Insulin; D011993:Recombinant Fusion Proteins