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I. Rowles et al. / Tetrahedron xxx (2016) 1e5
compared to the wild-type enzyme. This variant was used in
a preparative scale biotransformation resulting in a 94% conversion
4.3. Analysis of protein production and purity
to 4-bromo-
L
-phenylalanine (>99% ee).
Protein expression and purity was assessed using pre-cast
10e20 % TriseHCl polyacrylamide gradient gels (Bio-Rad, Hercu-
les, CA) and pre-stained broad range molecular weight marker
(NEB, Ipswich, MA). Electrophoresis of samples was at 80 V for 1 h
following the dye front and the gels stained with EZBlueÔ Gel
Staining Reagent.
Protein samples separated by SDS-PAGE were blotted onto
a PVDF membrane using a BioRad semi-dry blotter at 150 mA for
1 h. The membrane was washed with PBS for 15 min, the buffer
changed every 5 min, blocked with a milk solution (1 g milk
powder in 20 mL PBS). The membrane was washed with PBS, and
then incubated with an Anti-His-HRP conjugate antibody for 2 h.
The membrane was washed again then stained with a DAB/H2O2
solution.
4. Experimental
Competent cells (BL21 (DE3)) were purchased from Invitrogen
or Agilent and were transformed according to the manufacturer’s
protocol. The empty vector (pET-16b) originates from Novagen. LB-
Broth Miller and ampicillin were purchased from Formedium. All
other chemicals and reagents were purchased from Alfa Aesar,
Fisher or SigmaeAldrich.
Reverse phase HPLC was performed on an Agilent system
equipped with a G1379A degasser, G1312A binary pump, a G1329
autosampler unit, a G1315B diode array detector and a G1316A
temperature controlled column compartment. Columns and con-
ditions are indicated for each compound separately. Liquid phase
assays for the determination of relative rates were recorded on
a Spectramax M2 plate reader from Molecular Devices. Protein
4.4. Homology model
A homology model of RgrPAL was constructed, containing four
monomers, based on the PAL enzyme from R. glutinis (PDB acces-
sion code 1T6J) which has 71.9% sequence identity. Loops for which
there was no structural information and the three amino acids
constituting the MIO cofactor (Ala-Ser-Gly) were excluded from the
model building process. The MIO cofactor was built into the model
by transferring the cofactor from the template, followed by energy
minimisation of the cofactor. Homology models and docking were
performed with Accelrys Discovery Studio 3.1.
€
purification was performed on GE Healthcare AKTA Explorer 100
system using HiTrap Chelating HP columns (1 mL, GE Healthcare).
4.1. Production of PAL
A single colony of BL21 (DE3) pET 16b PAL was used to inoculate
8 mL of LB containing ampicillin (100
monitored and when a value of 0.6 was reached 6 mL of the starter
culture was used to inoculate 600 mL LB medium in a 2 L baffled
flask. The PAL protein was produced by incubating this culture for
18 h at 26 ꢁC with 250 rpm continuous shaking.
mg/mL), the OD600 was
4.5. Saturation mutagenesis
The homology model of PAL was used for the semi-rational re-
design of the PAL active site in an attempt to modify the substrate
profile of the enzyme. From the model potential sites for the mu-
tations were selected and these sites were: 143/144 (A), 272/276
(B), 369/372 (C), 473/477 (D) and 501/504 (E). Saturation muta-
genesis libraries were created at these positions and screened for
activity towards the substrates. The primers used for this were as
follows (see Table 5):
After growth the cells were harvested by centrifugation at
7000 rpm for 20 min, the cell pellet was re-suspended in 15 mL
0.1 M potassium phosphate buffer pH 7.7, transferred to 50 mL
falcon tubes and spun down at 4000 rpm for 20 min. The cell pellet
was stored at ꢂ20 ꢁC until use.
4.2. Protein purification
Table 5
2.5 grams of cell paste was defrosted and resuspended in 6 mL of
buffer A (100 mM KPi, pH 7.7, 300 mM NaCl). 6 mg of lysozyme was
added and the suspension was incubated at 37 ꢁC for 30 min, cooled
on ice and sonicated to disrupt the cell walls (20 s pulse; 25 s pause;
20 cycles; 12e15 kHz). Cell debris was removed by centrifugation
Primer sequences 50 to 30, only the forward primer sequence is shown
Library
Sequence
A
B
C
D
E
GCTCATCGAGNRTNRTCTCTGCGGCGTGACG
CGAAGGAGGGTNRTGGTCTGGTCNRTGGAACGGCCGTC
CCAGGACCGCNRTCCGCTCNRTACGTCGCCTCAGTTCC
GCTCAACTATCACGGCNRTGGCTTGGACNRTCACATCGCTGCTTACGC
CGTCCAGCCCGCANRTATGGGTNRTCAGGCCGTCAACTCG
(20,000 rpm, 25 min), the supernatant was filtered (0.45 mM sy-
ringe filter) and loaded on a pre-prepared HiTrap column. Before
loading of the supernatant the column was prepped by flushing it
with the following: 5 mL of filtered water; 1 mL of 0.1 M NiSO4;
5 mL of filtered water; 5 mL buffer A. Next the protein was eluted on
the AKTA using a stepwise gradient of buffer A and buffer B
(100 mM K-Pi, pH 7.7, 300 mM NaCl, 1 M imidazole) collecting 1 mL
fractions. The stepwise gradient for elution of the PAL protein was:
1) 10 mL 100% Buffer A, 2) 10 mL 80:20 buffer A:B, 3) 15 mL 65:35
buffer A:B. The wells containing the purified enzyme were collected
and concentrated to 1.5 mL using a Vivaspin column (30,000 MW
cut-off), the volume was adjusted to 2.5 mL with 0.1 M KPi pH 7.7
and the solution was desalted on a PD10 column (GE Healthcare),
equilibrated with 25 mL of 0.1 M KPi pH 7.7, and eluted with 3.5 mL
of 0.1 M KPi pH 7.7).
A QuikChange (Agilent) PCR reaction was carried out with pET
16b PAL as the template DNA using the primers stated above. PCR
was carried out with an initial melting at 95 ꢁC for 3 min then 21
cycles with a 95 ꢁC melting temperature for 1.5 min, a 55 ꢁC
annealing temperature for 3 min and a 68 ꢁC elongation tempera-
ture for 10 min and a final 68 ꢁC elongation step of 12 min. After the
PCR the parental strand of DNA was digested with 1
mL of DpnI for
3 h at 37 ꢁC, then 1
m
L of the digested PCR reaction mixture was
used to transform Escherichia coli XL-1 Blue chemically competent
cells (Agilent), according to the manufacturer’s protocol.
Eight colonies were picked and the plasmid DNA purified and
sequenced to confirm mutagenesis. The remaining colonies were
taken up in 1 mL of LB Broth and the plasmid library purified,1 mL of
this purified plasmid library was used to transform BL21 (DE3)
(Invitrogen).
The purity of combined and de-salted protein fractions was then
checked by SDS-PAGE (Fig. 1), after purification between 20 and
25 mg of protein was obtained per litre culture. Protein concen-
tration and yield was calculated using the BCA assay (Pierce)
according to the manufacturer protocol.
Rowles, Ian
