L.T. Quertinmont, S. Lutz / Tetrahedron xxx (2016) 1e6
5
biocatalysts. Our present study used small, focused libraries to
systematically probe the functional consequences of specific res-
idues and regions in OYE1. In contrast to similar previous studies,
the application of RAPPER enables faster and more efficient, semi-
quantitative evaluation of enzyme variants. Beyond changes to
individual amino acid positions, fragment overlap extension PCR
has successfully been applied to generate gene templates for ex-
ploring functional consequences of residual InDels (insertions/
deletions). Furthermore, RAPPER’s capacity to support parallel
screening of engineered biocatalysts with multiple substrates
enables a more comprehensive assessment of impact and contri-
butions of individual amino acid substitutions. Our present results
for OYE1 highlights the potential benefits of substrate profiling in
protein engineering studies, averting the risk of misleading results
from usage of a model substrate. Functional gains of amino acid
replacements such as P295A or changes in stereoselectivity ob-
served for W116I show significant substrate dependence. Similar
to other high-throughput screening platforms, RAPPER does have
limitations due to the small sample sizes. Protein yields for en-
supernatant containing fragment DNA was collected. For the sec-
ond PCR to reassemble fragments into the full-length genes, ali-
quots of the fragment DNA were mixed with universal flanking
0
primers (same sequence as for the first PCR but without 5 -tags).
Full-length PCR products were purified using the QIAgenPCR Pu-
rification kits (Qiagen, Valencia, CA) and quantified by UV absor-
bance spectroscopy at 260 nm.
4.3. In vitro transcription/translation of linear DNA
Linear DNA (PCR products) were used as templates for protein
expression of selected OYE variants to assess their catalytic activi-
23
ties as described previously. Briefly, protein synthesis was per-
formed by in vitro transcription/translation (IVTT), using the
PURExpress system and reaction products were directly tested for
ene-reductase activity with selected substrates of interest. With
each set of experiments, wild type OYE1 was run as a positive
control while dihydrofolate reductase (provided by NEB) served as
negative control. Typically, protein synthesis was terminated after
zyme variants from 10-
m
l PURE reactions are to low to assess
ꢁ
2
.5 h incubation at 37 C by storing the mixture on ice.
questions of expression level, foldability and stability of individual
candidates in detail. Furthermore, amino acid changes may result
in altered inhibitory properties and substrate binding affinity
which can affect the outcome of the screening assay and com-
plicate direct comparison of variants. Lead candidates are there-
fore routinely evaluated in follow-up studies using traditional
biochemical and biophysical methods and these results have
validated the semi-quantitative nature of RAPPER data, making
the protocol useful for initial screening of enzyme libraries. In
summary, the use of cell-free systems offers a simple, versatile
and very effective tool to accelerate the discovery process for
tailored biocatalysts.
4
.4. Large-scale protein expression and purification
To obtain larger quantities of purified OYE variants, PCR prod-
ucts were cloned into pET-14b (Novagen) via NcoI/XhoI restriction
sites and the plasmid DNA transformed into E. coli BL21(DE3) pLysS
cells. Individual colonies were grown overnight in 5 mL of 2YT
media containing ampicillin (100
overnight culture was then used to inoculate 250 mL of 2YT con-
taining ampicillin (100
the culture reached an OD(600) of 0.7, heterologous protein ex-
pression was induced by addition of IPTG (final concentration:
0
4
ꢁ
m
g/mL) at 37 C. An aliquot of the
ꢁ
mg/mL). Following incubation at 37 C until
ꢁ
.4 mM) for 18 h at 20 C. Cells were pelleted by centrifugation at
4
4
. Experimental
ꢁ
C, 4000 g for 20 min, the clear supernatant removed and pellets
ꢁ
stored at ꢂ20 C until further usage.
.1. General information
For protein purification, a cell pellet was resuspended in 24 mL
of buffer A (40 mM TriseHCl (pH 8.0), 20 mM NaCl) and mixed with
The PURExpressÒ kit was purchased from New England Biolabs
200 mL protease inhibitor cocktail (Sigma) and 20 mL of benzonase
(
Ipswitch, MA). All primers used were synthesized by Integrated
(Novagen). After 30 min storage on ice, cells were lysed by soni-
DNA Technologies (Coralville, IA). All other reagents were pur-
chased from Sigma Aldrich (St. Louis, MO) unless otherwise spec-
ified. GC spectra were obtained on an Agilent Technologies 6850 GC
ꢁ
cation. The suspension was centrifuged at 4 C, 10,000 g for 30 min
and the clear supernatant was loaded on a HiTrap Q FF (5 mL)
anion-exchange column, pre-equilibrated in buffer A. The resin was
washed with two column volumes (CVs) buffer A, followed by
a linear gradient over 10 CVs to 100% buffer B (40 mM TriseHCl (pH
8.0), 1 M NaCl). Protein elusion was monitored by UV absorption
instrument equipped with
a
chiral CycloSil-B column
(
30 mꢀ0.32 mm/0.25
m
m, Agilent, Santa Clara, CA) using hydrogen
as a carrier gas (flow rate 1.8 mL/min) and an FID detector (detector
temperature 200 C, split ratio 25:1).
ꢁ
(
280/460 nm) and product fractions were collected, followed by
concentration and buffer-exchange into buffer C (40 mM TriseHCl
pH 8.0), 300 mM NaCl) using a Millipore filter unit (MWCO:
0 kDa). The protein sample was further purified via size exclusion
4
.2. Creating linear templates of OYE variants
(
1
All mutations were prepared by a two-step fragment overlap
21,23
chromatography (Superdex 200, 10/300 GL column) in buffer C.
Product fractions were pooled, concentrated as described above,
and analyzed by SDS-PAGE.
extension PCR protocol using pET14b-OYE1 as a template.
Mutagenic primer pairs are listed in Table S1 and were used in
0
0
combination with universal flanking primers carrying a 5 -des-
thiobiotin-tag for gel-free fragment purification (forward: 5 -des-
0
0
thiobiotin-CTCGATCCCGCGAAA TTAATACGACT-3 ; reverse: 5 -
4.5. Enzymatic activity assay for OYE1 variants
0
desthiobiotin-CAGCAAAAAACCCCTCAAGACCCG-3 ). Following the
first PCR, reaction mixtures were diluted with an equal volume of
distilled water and added to high-capacity streptavidin agarose
beads (Pierce, Grand Island, NY) pre-equilibrated in binding buffer
Ene-reductase activity assays were performed at ambient tem-
perature under anaerobic conditions (Coy Laboratory, Grass Lake,
MI). For preparing reaction stock solution, individual substrates
(
5 mM TriseHCl (pH 7.5), 0.5 mM EDTA, 1 mM NaCl). After in-
(see below) were dissolved in 50 mM TriseHCl (pH 7.5), supple-
ꢁ
þ
cubation at 21 C for 15 min, the mixture was centrifuged at
mented with 200
mM NADP , 100 mM glucose, and glucose de-
1
3,000 rpm for 3 min and the supernatant discarded. The
hydrogenase (GDH) from Thermoplasma acidophilum (2 units for
IVTT reactions, 5 units for purified enzyme reactions). Individual
substrate concentrations were chosen to ensure vmax conditions or
maximum solubility. At these substrate levels, reaction times were
adjusted for 10e50% substrate conversion.
streptavidin-bound fragments were washed three times with
1
00
m
L binding buffer, followed by incubation in 50
mL elution
ꢁ
buffer (4 mM biotin, 10 mM TriseHCl, pH 8.5) at 21 C for 10 min.
The mixture was centrifuged at 13,000 rpm for 3 min and the clear