Download Supplementary Information

Supplementary Information
A stereospecific solid phase screening assay expressing both
(R)- and (S)-selective ω-aminotransferase.
Simon C. Willies,a James L.Galman, a Iustina Slabua and Nicholas J. Turner a
Plasmids, chemicals and strains
All chemicals were reagent grade, purchased from Sigma-Aldrich (St Louis, MO, USA) and
Fisher Scientific (Pittsburg, PA, USA), unless otherwise noted. Restriction enzymes, T4
ligase, and broad protein marker (2-212 kDa) E. coli DH5α and E. coli BL21 Star (DE3) were
purchased from New England Biolabs (Ipswich, MA, USA). Primers were purchased from
Eurofins MWG operon (Ebersberg, Germany) and Ni-NTA agarose were purchased from
Invitrogen (Carlsbad, CA) and pET21a(+) (Novagen, Germany) were used for gene
expression. The D-AAO gene was codon optimised for expression in E. coli and synthesised
by Geneart AG with the restriction sites NcoI and NotI at the 5’ and 3’ ends respectively in
the vector pMA. The D-AAO was excised from the vector using NcoI and NotI and inserted
into pRSF-Duet using the same restriction sites, to create pRSF-D-AAO. The AlaR gene was
cloned into pET16b as described elsewhere.1 The ATA-1172 and CV-ATA3 genes were
obtained from Prof. Wolfgang Kroutil (University of Graz) in pET21a. Aminotransferase
plasmid DNA (pLE1A17-ATA-50) was obtained from c-LEcta GmbH (Leipzig, Germany).
Subcloning of wild-type aminotransferase (pLE1A17-ATA-50)
The DNA sequence encoding aminotransferase (pLE1A17-ATA-50) was kindly provided by
c-LEcta and was subcloned into a compatible vector suitable for heterologous protein
expression in E.coli cells. The DNA insert and the vector backbone were assembled into
DNA multimers via POE-PCR as described elsewhere.4 The DNA multimers were
transformed into supercompetent E. coli DH5α cells and yielded pET21a-ATA-50. The
plasmid was sequenced and verified by GATC Biotech AG (Konstanz, Germany).
Cotransformation of D-AAO and ATA plasmids in E.coli BL21 (DE3) cells.
Plasmids (D-AAO and ATA) were cotransformed in E. coli BL21 (DE3) cells and plated onto
a Hybond-N membrane laid on LB/agar containing ampicillin (100 mg/mL) and kanamycin
(50 mg/mL) and incubated at 30 °C overnight.
The membrane was then transferred onto
fresh LB/agar containing IPTG (0.2 mM) ampicillin (100 µg/mL) and kanamycin (50
µg/mL), and incubated for a further 18 h at 20 °C. The membrane was then placed at -20 °C
overnight. To confirm the presence of the plasmids in E.coli cells, synthetic oligonucleotide
primers were designed to anneal to the complementary DNA strand of pET21a-CV-ATA and
pRSF-DAAO genes as an example.
DAAO Rev: CAG ATT CGG ACG GGT CAG TGC (P1)
DAAO Fw: GCC AAA ATT GTT GTT ATT GGT GC (P2)
CV-ATA Fw: AGC GGC CAG AAA CAG CGT ACC ACC TC (P3)
CV-ATA Rev: GGC CAG ACC ACG TGC TTT CAG (P4)
10 colonies were picked and a ‘colony PCR’ was performed using taq polymerase (NEB)
(Table 1). All selected colonies confirmed the presence of both genes in the cell Fig.1.
Cycle step
Temp.
Time
Cycles
Initial denaturation
95 °C
5 mins
1
Denaturation
Annealing1
Extension2
95 °C
58 °C
68 °C
30 s
30 s
2 mins
30 cycles
Final extension
72 °C
4 °C
10 min
hold
1
Table 1: PCR cycles for colony PCR
Fig 1: DNA gel of genes amplified in colony PCR; A) DNA ladder, B) CV-ATA gene
amplification only using primers (P3/P4), C) DAAO gene amplification only using primers
(P1/P2), D) Simultaneous CV-ATA and DAAO amplification using (P1/P2/P3/P4) in PCR
mix.
Coexpression and purification of heterologous protein DAAO and ATA
The freshly-prepared strain E. coli BL21 (DE3) containing pET21a-ATA and pRSF-DAAO
was cultivated in 600 mL of the LB medium supplemented with 50 µg/mL kanamycin and
100 µg/mL ampicillin in 1-L Erlenmeyer flasks at a rotary shaking rate of 250 rpm at 37 °C.
The
recombinant
protein
expression
was
induced
by adding
isopropyl
β-D-1-
thiogalactopyranoside (IPTG) (0.2 mM, final) when A600 reached 0.6 - 0.8. The cell cultures
were incubated at 18 °C for 16 h. The cells were harvested by centrifugation at 4 °C, and
resuspended in 20 mL of TRIS-HCl (pH 7.4) with 100 mM NaCl and 50 µM PLP. The cell
pellets were lysed in an iced bath by ultra-sonication by Fisher Scientific Sonic
Dismembrator Model 500 (3-s pulse, total 90 s, at 30% amplitude). After centrifugation at
4,000 rpm for 20 min, the lysate supernatant was loaded on a 20 mL column with a capped
bottom outlet (purchased from BioRad, Munich) containing 1 mL of the 50 % Ni-NTA
slurry. A 10 mL wash buffer containing 50 mM TRIS, 30 mM imidazole and 50 uM PLP at
pH 7.4 was used to elute unbound proteins. The his-tag ATA-50 protein was eluted with 10
mL elution buffer containing 50mM TRIS, 300 mM imidazole, 100 mM NaCl and 50 uM
PLP at pH 7.4 and then concentrated in a micropore filter. The fractions of unbound proteins
from the flow-through and washing steps were collected and passed through a preequilibrated 1 mL strep-tactin column (IBA, Göttingen) and subsequently washed with 10
mL wash buffer containing 100 mM Tris, 150 mM NaCl, 1 mM EDTA at pH 8.0. The streptag D-AAO was eluted with the aforementioned buffered solution with additional 2.5 mM
desthiobiotin and the purity of both purified proteins was analysed by SDS-PAGE which
gave more than 95% purity (Figure 2).
Figure 2: SDS gel of pRSF-DAAO and coexpressed pET21a-ATA-50 (gene provided by cLEcta) with pRSF-DAAO
Solid phase screening for R-selective colonies
A pre-screen solution containing horse radish peroxidase (0.1 mg/mL), pyruvate (0.2 mg/mL)
and 50 mM potassium phosphate pH 8.0 was pre-soaked onto filter paper. The membrane
was laid onto the filter paper and left at room temperature for 1 hour. The membrane was
freeze-thawed with liquid nitrogen then transferred to a pre-soaked filter paper containing
colorimetric assay solution: 1 x 3,3’-diaminobenzidine tablet (Sigma), 10mM substrate,
horseradish peroxidase (0.1 mg/mL, Sigma) in 50 mM potassium phosphate pH 8.0. The
membrane was left at room temperature and colour change is observed over time.
Solid phase screening for S -selective colonies
The (S)-selective solid phase screen is analogous to the (R)-selective screen with an
additional prepared membrane containing alanine racemase (AlaR). A 5mL starter culture of
Ala-pET16b in E. coli BL21 (DE3) was grown in LB media at 37 °C for 4 hours (OD ≈0.3).
300 μL of starter culture was plated onto an ampicillin (100 mg/mL) LB agar plate and grown
at 30 °C overnight. The AlaR membrane was transferred onto fresh LB agar containing
IPTG (0.2 mM) and ampicillin (100 mg/mL) and grown at 25 °C for four hours. The
membrane was removed and freeze-thawed with liquid nitrogen. The membrane containing
ATA-50/D-AAO colonies was laid onto AlaR membrane to produce a ‘sandwich’. The
sandwich membranes are then transferred to the pre-screened and colorimetric assay soaked
filter paper respectively. Hits were picked and sequenced to identify variants obtained.
Library generation
Mutagenesis libraries were generated using the Stratagene Quick-change™ site directed
mutagenesis kit (stratagene). An NNS library was created in ATA-117 at amino acid position
Phe116 (Figure 3) using the primers given in (Table 2). PCR conditions were as per the
manufacturer’s instructions and parental DNA was removed by DpnI digestion for 2 hours at
37 °C. The PCR product was then transformed in E. coli XL-1 Blue, and 10 random colonies
from the library were picked for sequencing to confirm library quality. All cells on the plate
were resuspended in 5mL LB, centrifuged and DNA extracted to provide the mutant library.
Primer
Sequence
ArR_116NNS_For
AAA ACC GAA CTG CGT GAA GCA NNS GTT AGC GTT AGC
ATT ACC CGT
ArR_116NNS_Rev
ACG GGT AAT GCT AAC GCT AAC SNN TGC TTC ACG CAG
TTC GGT TTT
Table 2. Mutagenic PCR Primers
Figure 3. Enzyme binding site of ATA-117
Liquid phase assay
A stock assay solution containing 10U D-AAO, 1 mg HRP, 2 mg pyruvate, 3mg 4aminoantipyrine (AAP), 2 mg 2,4,6-tribromo-3-hydroxybenzoic acid (TBHBA) all made up
to 10 mL in 100mM HEPES pH8.0. Amine substrates were made to 200 mM in DMSO.
Assays were carried out in 96 well MTP with 10 µL substrate/DMSO, 95 µL assay solution
and 95 µL enzyme solution. Assays were monitored on a spectrophotometer at 510 nm and
30 °C for 30 minutes.
160
140
% Relative Activity
120
100
R-MBA
80
R-EBA
60
R-PBA
40
20
0
ArR116W
_________
1.
S. C. Willies, J. L. White, and N. J. Turner, Tetrahedron, 2012, 68, 7564–7567.
2.
C. K. Savile, J. M. Janey, E. C. Mundorff, J. C. Moore, S. Tam, W. R. Jarvis, J. C.
Colbeck, A. Krebber, F. J. Fleitz, J. Brands, P. N. Devine, G. W. Huisman, and G. J.
Hughes, Science, 2010, 329, 305–9.
3.
U. Kaulmann, K. Smithies, M. E. B. Smith, H. C. Hailes, and J. M. Ward, Enzyme
Microb. Technol., 2007, 41, 628–637.
4.
C. You, X.-Z. Zhang, and Y.-H. P. Zhang, Appl. Environ. Microbiol., 2012, 78, 1593–
5.