Cloning, expression, purification, and characterization of a designer protein with repetitive sequences.
An artificial protein containing alternating hydrophilic-hydrophobic blocks of amino acids was designed in order to mimic the structure of synthetic multiblock copolymers. The hydrophobic block consisted of the six amino acids Ala Ile Leu Leu Ile Ile (AILLII) and the hydrophilic block of the eight amino acids Thr Ser Glu Asp Asp Asn Asn Gln (TSEDDNNQ). The coding DNA sequence of the cluster was inserted into an commercial pET 30a(+) vector using a two step strategy. The expression of the artificial protein in Escherichia coli was optimized using a temperature shift strategy. Only at cultivation temperature of 24 degrees C after induction expression was observed, whereas at 30 and 37 degrees C no target protein could be detected. Cells obtained from a 15L bioreactor cultivation of E. coli were disintegrated by mechanical methods. Interestingly, glass bead milling and high pressure homogenization resulted in a different solubility of the target protein. The further purification was carried out by affinity chromatography using the soluble homogenized protein. Extreme conditions (6M urea, 0.5M NaCl) were applied in order to prevent aggregation to insoluble particles. The designer protein showed an extremely high tendency to form dimers or trimers caused by intermolecular interactions which were even not broken under the conditions of SDS-polyacrylamide gel electrophoresis, rendering the behavior during purification different from proteins usually found in nature. The protein preparation was not completely pure according to SDS-PAGE stained by Coomassie blue or silver. In MALDI-TOF-MS, nano-ESI qTOF-MS of the entire protein preparation and nano-ESI-MS after digestion by trypsin and chymotrypsin impurities were not detectable.
Gerber
Silke
S
Department of Pharmacy, Faculty of Natural Sciences I, Martin-Luther-University Halle-Wittenberg, Biozentrum, Weinbergweg 22, 06099 Halle (Saale), Germany.
Kirchhof
Kristin
K
Kressler
Jörg
J
Schmelzer
Christian E H
CE
Scholz
Carmen
C
Hertel
Thomas C
TC
Pietzsch
Markus
M
D000595:Amino Acid Sequence; D019149:Bioreactors; D018929:Cell Culture Techniques; D002458:Cell Fractionation; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D020449:Repetitive Sequences, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011994:Recombinant Proteins