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Hummel (University of Dꢁsseldorf, Germany). The CHMO gene
Cloning
Cloning of the nemA gene into pET22b(+) was performed applying
the FastCloning method so that the C-terminal His-tag was in
frame. Primers for amplification of the gene from genomic DNA:
(
GenBank: BAA86293.1) was cloned readily into pET28b(+). Trans-
formations of E. coli strains with the plasmids were performed with
[27]
[24]
the heat shock method as described by Chung et al.
5
5
’-CGG CCG CAA GCT TAG CCT GCT TCA GG-3’,
Genetic methods and sequence analysis
’-CGG AAT TAA TTC GGA TCC ATG AAA CTC TTG CAA CCG C-3’.
Total genomic DNA from P. putida NCIMB 10007 was extracted by
using a DNeasy Blood and Tissue Kit from Qiagen (Hilden, Germa-
ny). Plasmid isolation (Analytik Jena, Jena, Germany or Fermentas,
St. Leon-Roth, Germany), PCR purification, and gel extraction
Primers for amplification of the plasmid pET22b(+):
5
5
’-GCG GTT GCA AGA GTT TCA TGG ATC CGA ATT AAT TCC G-3’,
’-CCT GAA GCA GGC TAA GCT TGC GGC CG-3’.
(
Qiagen, Hilden, Germany or Roche, Mannheim, Germany) as well
as QuikChange (Agilent Technologies, Santa Clara, CA, USA) muta-
genesis were performed according to the manufacturers’ instruc-
tions. The StrataClone PCR Cloning Kit from Agilent Technologies
The genes xenA and xenB were cloned into pGaston by using the
restriction enzymes NdeI and BamHI by addition of their recogni-
tion sites to gene-specific primers for amplification of the genes
from isolated genomic DNA:
(
Santa Clara, CA, USA) was used to subclone DNA fragments. DNA
sequencing was conducted by GATC (Konstanz, Germany) or Euro-
fins MWG Operon (Ebersberg, Germany), and analyses were per-
formed by using the software Geneious.
XenA: 5’-GGA ATT CCA TAT GTC CGC ACT GTT CGA ACC CTA
CAC-3’,
[25]
5
’-CCA ATT GGA TCC GCG ATA GCG CTC AAG CCA GTG C-3’;
XenB: 5’-GGG AAT TCC ATA TGA CCA CGC TTT TCG ATC CG-3’,
’-CGC GGA TCC CAA CCG CGG GTA ATC GAT GTA-3’.
Gene walking
5
Starting from sequence fragments obtained from P. putida (provid-
[26]
ed by Prof. Littlechild), a BLAST search was performed to identify
full gene lengths. The 3’-sequence of the xenB gene was identified
by using an oligonucleotide forward primer:
The gene fragment and the empty vector were digested, ligated,
and transformed into E. coli TOP10. In these constructs, XenA and
XenB contained a C-terminal His-tag to facilitate purification.
5
’-CCA TTG AAG TGT GGG GCG CGA AC-3’
and the 5’-sequence of the xenB gene was identified by using two
reverse primers:
Expression
Expression of XenA and XenB in E. coli was performed by inocula-
5
5
’-GTG AAC TGG GCA AGC GTG GCA TTG-3’,
’-CTG CAA CTG CCC ACC GCA TCA TC-3’.
tion of LB media (400 mL) supplemented with ampicillin
À1
(100 mgmL ) with an overnight culture (4 mL; 1:100). This was in-
cubated at 378C at 180 rpm until OD600 =0.6–0.8 was reached.
Then expression was induced with 0.2% (w/v) l-rhamnose, and in-
cubation was continued at 258C (XenB) or 308C (XenA) for 8 h.
The cultivation of NemA was performed in the same way except
for the used medium (TB), the inductor (1 mm IPTG), and the ex-
pression conditions (at 258C for 4 h). CHMO and LK-ADH were also
After initial denaturation for 4 min at 958C, the cycling program
was followed for 30 cycles: 30 s, 958C denaturation; 30 s, 558C
primer annealing; 3 min, 728C elongation; unspecific annealing of
the primer: 30 s, 958C denaturation; 30 s, 408C primer annealing;
3
3
3
min, 728C elongation; and complementary strand synthesis for
0 cycles: 30 s, 958C denaturation; 30 s, 608C primer annealing;
min, 728C elongation. The final elongation step was performed
À1
cultivated in TB supplied with 50 mgmL kanamycin (CHMO) or
[22]
ampicillin (LK-ADH) as described previously. Expression was in-
over 15 min at 728C. The DNA fragments obtained were subcloned
by using the StrataClone PCR Cloning Kit, transformed into E. coli
TOP10 cells, and colonies were examined for the presence of in-
serts in a colony PCR with M13 primers:
duced with 0.1 mm IPTG followed by cultivation for 16 h at 258C.
Cells were harvested by centrifugation at 4500ꢂg for 20 min at
4
8C.
forward: 5’-TGT AAA ACG ACG GCC AGT-3’,
reverse: 5’-CAG GAA ACA GCT ATG ACC-3’.
Enzyme purification
For cell disruption, the cell pellet was resuspended in 25 mL buffer
After initial denaturation for 10 min at 958C, the cycling program
was followed for 25 cycles: 30 s, 958C denaturation; 30 s, 568C
primer annealing; 3 min, 728C elongation. The final elongation
step was performed over 10 min at 728C. Plasmids of colonies,
which contained DNA fragments, were subsequently isolated and
sequenced. The xenB gene is 1050 bp long (GenBank code:
KF055345). The nucleotide sequence of a third contig showed high
homology to the C terminus of a putative N-ethylmaleimide reduc-
tase. To elucidate the missing N terminus, the forward primers:
(
3
sodium phosphate buffer, 100 mm, pH 7.5 supplemented with
00 mm NaCl) that included 30 mm imidazole. Cell disruption was
performed by a single passage through a French pressure cell at
000 psi. By centrifugation at 9000ꢂg for 20 min, cell debris was
2
separated from the crude cell extract. Purification was performed
by affinity chromatography by the C-terminal His-Tag with an auto-
mated ꢃkta purifier system. A 5 mL HisTrap FF column was equili-
brated with buffer. After the crude cell extract was applied on the
column, unbound protein was eluted with five column volumes of
buffer supplied with 39 mm of imidazole. The elution of the de-
sired ERED was accomplished by three column volumes of buffer
that contained 300 mm imidazole. Washing, flow through, and elu-
tion fractions were analyzed by SDS-PAGE. Desired fractions were
collected for desalting by dialysis (XenA and XenB) in dialysis tubes
5
5
’-CAA TAC GTG CGC GGT TTT CGA CG-3’,
’-CCT GGT CTT CGC CAT TGT CCA CG-3’
were used following the procedure given above. The GenBank
code is KF744040.
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemCatChem 2014, 6, 1021 – 1027 1025