High level expression of alpha-human atrial natriuretic factor as a fusion polypeptide with phage fr coat protein in Escherichia coli.
A synthetic DNA sequence coding for the 28 amino acid residues of alpha-human atrial natriuretic factor (alpha-hANF) and the N-terminal linker tripeptide Ile-Asp-Lys was inserted in the 3'-terminal part of the RNA bacteriophage fr coat protein gene. The cloned hybrid gene was isolated and placed into an expression vector under the control of the inducible E. coli tryptophan promoter and phage fr coat protein translation initiation region (TIR) sequence. In an appropriate host strain the expressed fusion protein accounts for at least 10% of the total cellular protein. In order to achieve high-cell density in a bioreactor while maintaining efficiency of alpha-hANF expression, improved cultivation conditions were selected using modified Shielach-Bauer's culture media containing glucose, yeast extract and bacto tryptone at an initial concentration of 2 g l-1 of each, adding concentrate of medium throughout the microbial growth and maintaining the dissolved oxygen in a range of 25-30%. At 13-14 h cultivation, the cell density reached 40 g cell dry weight per liter and the yield of fusion protein exceeded 45 mg g-1 cell dry weight. Fusion protein from solubilized E. coli cells was purified to homogeneity by ion exchange chromatography on DEAE-, CM-cellulose, QAE Sephadex A25 columns and selective precipitation.
Berzins
V
V
Institute of Molecular Biology, University of Lativia, Riga.
Jansone
I
I
Skangals
A
A
Kalnins
P
P
Liepa
S
S
Baumanis
V
V
D000595:Amino Acid Sequence; D009320:Atrial Natriuretic Factor / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002213:Capsid / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification
C472955:NPPA protein, human; D011993:Recombinant Fusion Proteins; D009320:Atrial Natriuretic Factor; D004247:DNA