Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress promoters uspA and uspB.
The use of the uspA and uspB promoters (universal stress promoters) for heterologous protein production in Escherichia coli is described. Best results were obtained with a moderate copy number vector (15-60 copies) bearing the uspA promoter, reaching 4.6 mg/g dry cell weight (DCW) of ZZ-proinsulin secreted to the periplasm and 1.9 mg/g DCW secreted to the culture medium. These values are about 1.7-fold higher than those previously reported with the same ZZ fusion tag and the SpA leader peptide showing that these stress promoters are potentially valuable for recombinant protein secretion in E. coli. It is further demonstrated that the use of M9 minimal medium is advantageous for protein secretion as compared to LB rich medium.
Mergulhão
F J M
FJ
Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal.
Monteiro
G A
GA
Larsson
G
G
Sandén
A M
AM
Farewell
A
A
Nystrom
T
T
Cabral
J M S
JM
Taipa
M A
MA
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics*; D018628:Gene Dosage; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011384:Proinsulin / Q000096:biosynthesis* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D001426:Bacterial Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C116144:UspB protein, E coli; C078564:universal stress protein A, Bacteria; D011384:Proinsulin