Erythromycin esterase type II from Escherichia coli that degrades macrolid antibiotics. Part is RFC[25] compatible and is flanked by RFC[25] pre- and suffix.
Usage and Biology
Protein Data Table for the erythromycin esterase (EreB) BBa_K1159000
Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein) ATGGCCGGCAGGTTCGAA ... GTTTATGAAACCGGT ORF from nucleotide position -8 to 1260 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
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Production and purification of recombinant EreB
Figure A: Streptavidin affinity chromatography for the erythromycin esterase
Figure B: Analytical size exclusion chromatography on a Superdex 200 10/30 column
Figure C: Preparative size exclusion chromatography on a Superdex 75 16/60 column
Figure D: SDS-gel of recombinant EreB with the marker (M) followed by the concentrated throughput of the streptavidin affinity column and 6 fractions collected from the elution peak
The recombinant production and purification was carried out twice, in a first attempt 2 L of LB-media were used for an analytical purpose whereas in the second attempt we produced enough purified enzyme for all subsequent experiments. This preparation was carried out in 6 x 2L of LB media. Protein production was in both cases induced at OD = 0.8 by adjusting the cell culture to 5 mM of arabinose and was carried out for 4 h for the first and 5 h for the second preparation. Cell disruption was performed by ultrasonic sound in both cases. The cell lysate was then dialyzed against 5 L of SA-buffer and subsequently applied to streptavidin affinity columns. After the application of the protein, the column was washed with SA-buffer until a base line was reached. Afterwards the protein was eluted using 5 mM biotin. During the first preparation 2-mercapto-ethanol was added after the chromatographic steps. In order to avoid oxidation of cysteine residues to disulphid-bridges, which is not desired for the cytosolic EreB protein, the preparative purification was carried out with buffers containing 5 mM of 2-mercapto-ethanol in all buffers. When comparing the size exclusion chromatograms, obtained from the analytical and the preparative purification, it can be stated that there is still a considerable aggregation peak near the void volume (Fig. B) of the column in the first attempt, which was nearly not the case for the preparative preparation (Fig. C). Therefore we would give the advise to use strictly reducing conditions while working with recombinant EreB. The finally resulting yields of the preparative purification have been determined by absorption measurement of the aromatic amino acids at 280. The total yield was determined to 25 mg of pure protein which is 2.1 mg/L of LB culture.
Sequence and Features
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 189 Illegal XhoI site found at 599 Illegal XhoI site found at 1189
