G. Grogan et al.
(10 mL) with antibiotics (100 mgmLꢀ1 kanamycin, 34 mgmLꢀ1 chlor-
amphenicol). After overnight incubation at 378C with shaking, the
culture was used to inoculate M9 minimal medium (1 L plus
100 mgmLꢀ1 kanamycin and 34 mgmLꢀ1 chloramphenicol). When
the OD600 reached 0.8, protein expression was induced with IPTG
(1 mm), 5-ALA (1 mm), FeCl3 (0.5 mm) and riboflavin (5 mgLꢀ1). After
overnight incubation at 208C with shaking, the cells were harvest-
ed by centrifugation, resuspended in potassium phosphate buffer
(50 mm, pH 7.0, “buffer”) and disrupted by ultrasonication.
applicability of the vector to a sequence-diverse library of P450
genes.
Conclusions
A new platform for the rapid construction of redox-self-suffi-
cient P450s was established and validated through the expres-
sion of at least 13 soluble fusion proteins by using the versatile
reductase system from P450RhF. Detailed characterisation of
the purified chimeras P450cam-RhF-Red and XplAP450-RhF-
Red generated by using the LICRED platform indicated that
they were active, redox-self-sufficient monomers employing a
predominantly intramolecular electron-transfer chain with a
coupling efficiency that can exceed 80%. The system was read-
ily applied to the creation of families of chimeric P450s based
on sequence-diverse prokaryotic P450 haem domains. The ap-
plication of the LICRED platform to the expression, characteri-
sation and application of a variety of orphan eukaryotic P450s
from both mammals and plants is currently underway. This
new technology platform, combined with a rapid colorimetric
activity screening, represents a robust and cost-effective strat-
egy for identifying new P450 activities and generating redox
self-sufficient oxygenases for industrial biocatalysis.
After centrifugation, the crude supernatant was filtered and loaded
onto a nickel–agarose affinity column (5 mL, GE Healthcare). The
N-terminally His6-tagged proteins were eluted with an increasing
gradient of imidazole (30–500 mm) in buffer. Fractions containing
the fusion protein were concentrated and applied to a Superdex
200 (GE Healthcare) gel filtration column that had been pre-equili-
brated with the buffer. Protein purity was assessed by using SDS-
PAGE on a MiniProtean 3 system (BioRad). Protein samples were di-
luted in 4ꢁ SDS-PAGE loading buffer comprising glycerol (1 mL),
dH2O (2 mL), Tris-HCl (1 mL, 0.5m, pH 6.8), SDS (1.6 mL 10% w/v),
b-mercaptoethanol (0.4 mL) and bromophenol blue (20 mg). Sam-
ples were then boiled for 5 min at 1008C. The gels consisted of a
top layer of 4% (w/v) acrylamide stacking gel and a bottom layer
of 12% (w/v) acrylamide separating gel. The protein samples were
run at a constant voltage of 200 V.
Resting-cell biotransformations: Resting-cell assays were carried
out to assess the hydroxylation of (1R)-(+)-camphor by P450cam-
RhF-Red and the degradation of the explosive compound hexahy-
dro-1,3,5-trinitro-1,3,5-triazine (RDX) by XplAP450-RhF-Red. A single
colony of E. coli Rosetta 2 (DE3) (Novagen/Merck) transformed with
LICRED containing XplA haem domain or P450cam was used to
inoculate LB medium (5 mL plus 100 mgmLꢀ1 kanamycin and
34 mgmLꢀ1 chloramphenicol). After overnight incubation at 378C
with shaking, a culture (1 mL) was used to inoculate M9 minimal
medium (100 mL) with antibiotics (100 mgmLꢀ1 of kanamycin and
34 mgmLꢀ1 of chloramphenicol). When the OD600 reached 0.8, pro-
tein expression was induced with IPTG (1 mm), 5-ALA (1 mm), FeCl3
(0.5 mm) and riboflavin (5 mgLꢀ1). Cultures were incubated over-
night at 208C with shaking, the cells were harvested by centrifuga-
tion and a portion of the wet cell paste (300 mg) was resuspended
in potassium phosphate buffer (3 mL, 50 mm, pH 7.0) in a sterile
15 mL Falcon tube. d-Camphor and RDX were added to final con-
centrations of 10 mm and 150 mm, respectively, and the tubes were
put in a shaking incubator at 208C. Analysis of camphor hydroxyl-
ation was performed with an Agilent 5975C GC/MS. Helium was
used as the carrier gas at flow rate of 1.2 mLminꢀ1. The GC was
equipped with an Agilent HP5 column (30 m, 250 mm i.d., 0.25 mm
film thickness). The temperature program for camphor was: 1208C
for 3 min, then 120–1608C at 108Cminꢀ1. The temperatures were
2508C for the injection and for the detector. A sample of 1 mL was
injected onto the column. The retention times of camphor and 5-
exo-hydroxy camphor were 3.5 and 6.5 min, respectively. RDX deg-
radation was measured by HPLC, as previously described.[28]
Experimental Section
Design and construction of the LICRED vector: First, site-directed
mutagenesis was performed on the DNA encoding the reductase
domain of P450RhF by using overlap extension PCR to remove the
BseRI recognition site naturally present in the reductase gene
(CTCCTC!CTACTC). The sequence coding for the first five amino
acids of the linker was then modified in order to include the se-
quence for ligation-independent cloning and also the recognition
site for BseRI (GTG CTG CAC CGG CAT!CGC GCC TTC TCC TCA).
The LICRED plasmid was then constructed by ligating the modified
P450RhF reductase gene between the BseRI and NdeI restriction
sites in the pET-YSBLIC 3C plasmid.[24] The vector also included a
short sequence coding a hexahistidine tag at the N terminus of the
LIC site in order to facilitate further purification of the protein by
nickel affinity chromatography and its visualisation through West-
ern blotting analysis.
Cloning, overexpression and purification of the chimeric pro-
teins: Primers for each gene target encoding a P450 haem domain
were designed with standard 5’ LIC ends, for example, in the case
of P450cam-RhF-Red: P450cam_LIC_Forward (5’-CCA GGG ACC
AGC AAT GAC GAC TGA AAC CAT ACA AAG CAA CGC-3’) and
P450cam_LIC_Reverse (5’-GAG GAG AAG GCG CGT ACC GCT TTG
GTA GTC GCC GGA TC-3’). A full list of the primers used to create
the fusion proteins in this paper is available in the Supporting
Information. In each case, in order to obtain the fusion construct,
the stop codon in all haem-domain sequences was removed.
Size-exclusion chromatography–multiangle laser light scattering
(SEC–MALLS) analysis: The oligomeric state of the P450cam-RhF-
Red and XplAP450-RhF-Red fusion proteins was determined by
using SEC-MALLS measurements performed on a UFLC purifier
system (Shimadzu) connected to a UV/Vis detector (SPD-20 A, Shi-
madzu), a multiangle light-scattering detector (DAWN HELEOS II,
Wyatt Technology, Santa Barbara, CA, USA) and a refractive index
detector (OPTILAB rEX, Wyatt Technology). A SuperdexTM 200 (GE
Healthcare) gel filtration column was pre-equilibrated with buffer
at a flow rate of 0.5 mLminꢀ1. Sample volumes of 100 mL were
Genes encoding P450cam and XplAP450 were amplified in 50 mL
PCRs by using Phusion DNA polymerase (New England Biolabs)
under the following cycle parameters: one cycle of 988C for 5 min,
then 35 cycles at 988C for 10 s, 688C for 30 s and 728C for 60 s.
The PCR product was subcloned into the LICRED plasmid and
transformed into expression strains of E. coli, as previously de-
scribed for the pET-YSBLIC 3C vector.[24] Single colonies of trans-
formants were used to inoculate cultures of Luria–Bertani broth
992
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 987 – 994