FULL PAPER
were centrifuged and supernatants were discarded. Cell pel-
lets were washed with Tris-HCl (350 lL, pH 8.3) and resus-
pended in Tris-HCl (350 lL, pH 8.3) containing lysozyme
(0.5 mg/mL), deoxyribonuclease I (0.1 lg/mL), and MgCl2
(10 mM). After incubation at 37 °C for 45 minutes, the plates
were centrifuged and the lysate (150 lL) was transferred to a
96-well plate. Then 8-pnpane (150 lM) in DMSO (1%) was
added to the lysate and incubated at room temperature.
After 5 minutes, NADPH (1 mM) was added and the absor-
bance at 410 nm was measured with a microplate spectro-
photometer (SPECTRAmax, Molecular Devices).
Synthesis of p-Nitrophenoxyoctane (8-pnpane)
1-Bromooctane (1 g, 5.18 mmol) and 4-nitrophenol, sodium
salt (0.92 g, 5.71 mmol) were refluxed in DMSO (30 mL) at
120 °C for 5 hours. The DMSO was distilled off to near dry-
ness. The resulting brown residue was loaded onto a silica
column and eluted with 10:1 mixture of petroleum ether
1
and diethyl ether. The yield was 30%. H NMR (CDCl3): d =
8.18 (m, 2H), 6.93 (m, 2H), 4.04 (t, 2H), 1.81 (p, 2H), 1.33 (m,
10H), 0.89 (t, 3H); anal. calcd. for C14H21O3N: C, 66.91; H,
8.42; N, 5.57; found: C, 66.97; H, 8.34; N, 5.52.
CO binding: The most active clones identified from the
screen were cultured on a larger scale. Single colonies
were cultured at 37 °C overnight in LB (5 mL) containing
ampicillin (100 mg/mL). The overnight growth (0.5 mL)
was used to inoculate media containing TB (50 mL), ampi-
cillin (100 lg/mL), thiamine (5 lg/mL), and trace elements
(0.25 lL/mL). After the OD600 reached 0.6 ± 1.0, the tem-
perature was decreased to 30 °C, and enzyme expression
was induced by adding d-aminolevulinic acid hydrochlo-
ride (1 mM) and isopropyl-b-d-thiogalactopyranoside
(1 mM).
After 24 hours, the cells were harvested by centrifugation
and the supernatants discarded. The pellets were washed
with Tris-HCl (15 mL, pH 8.3). Cells were resuspended in
Tris-HCl (5 mL, pH 8.3), sonicated, and centrifuged. The
supernatants were further cleared through a 0.45 lM filter.
P450 BM-3 concentrations were measured from the CO-dif-
ference spectra.
Random Library Generation and Screening of
P450 BM-3
P450 BM-3 containing a His6 tag was amplified from pT-
USC1BM3 by PCR techniques using a proofreading polymer-
ase Pfu to introduce a BamHI upstream of the start codon
and an EcoRI site immediately downstream from the stop
codon. The two oligonucleotides used were: 5'-cgcggatc-
catcgatgcttaggaggtcatatgacaattaaagaaatgcctc-3' (BamHI site
underlined) and 5'-ccggaattcttaatgatgatgatgatgatgcccagcc-
cacacgtcttttgc-3' (EcoRI site underlined). The PCR product
was digested with BamHI and EcoRI. The P450 BM-3 gene
was ligated into expression vector pCWOri (+)[24]
(pBM3BamSacEco), which is under the control of double
Ptac promoter and contains an ampicillin resistance coding
region. A silent mutation was introduced to construct a SacI
site 130 bases upstream of the end of the heme domain. The
QuikChange (Stratagene) protocol was followed and the pri-
mers were: 5'-catacaaactacgagctcgatattaaagaaac-3' (SacI
site underlined) and 5'-gtttctttaatatcgagctcgtagtttgtatg-3'
(SacI site underlined).
Random mutagenesis: Mutagenic PCR was performed on
the heme domain in a 100 lL reaction volume as de-
scribed[18] with some modifications. The mutated P450 BM-
3 fragment was 1291 base pairs. The reaction contained
MgCl2 (7 mM), forward and reverse primer (40 pmol each,
5'-acaggatccatcgatgcttaggaggtcatatg-3', and 5'-gtgaaggaa-
taccgccaag-3'), pBM3BamSacEco (10 ng), dNTPs (0.2 mM
dGTP, 0.2 mM dATP, 1.0 mM dCTP, 1.0 mM dTTP), and Taq
polymerase (5 units, Roche), KCl (50 mM), and Tris-HCl
(10 mM, pH 8.3, 20 °C). MnCl2 (0.0, 0.05, and 0.1 mM) was
added to the PCR mixture to alter the error rate of the poly-
merase. PCR was performed in a thermocycler (PTC200, MJ
Research, Waltham, MA) for 30 cycles (95 °C, 45 s; 50 °C,
30 s; 72 °C, 2 min). The PCR product was restricted with
BamHI and SacI and ligated into expression vector pCWOri
(+). The resulting plasmid was transformed into E. coli
strain DH5a and the colonies were selected on agar plates
containing ampicillin (100 lg/mL).
Acknowledgements
We thank Yaniv Dubowski, Dr. Peter Green and Patrick Cir-
ino for their assistance with the GC/MS. This work is sup-
ported by U. S. National Science Foundation Grants DBI-
9807460 (ETF) and BES-9981770 (FHA).
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