method of PCR was used for the rapid and specific amplification of
DNA-fragments of interest, here pUC18 vectors with the fdx4-insert. Besides
restriction sites within the PCR-primer sequences were built in for later
cloning steps, too. The standard PCR protocol and the components for the PCR
reaction which were used, are shown in Table A1. Annealing temperature and
elongation time were adjusted based on primer characteristics and the product
size, respectively. PCR products were analyzed and separated by agarose gel
electrophoresis. These separated DNA fragments were excised and purified using
a PCR purification kit.
Agarose gel electrophoresis
gel electrophoresis was performed to separate DNA fragments according to their
size and therefore used to confirm each step of the cloning process. Agarose
gel was prepared by adding appropriate amount of agarose into TAE buffer,
samples were mixed with 6x loading dye including SYBR Green and the DNA
fragments were separated in an electric field (80-120 V).
agarose concentration of the gels was set to 1.0 % according to the size of DNA
fragments to be analyzed. A 1 kbp DNA marker was applied as a size standard.
finishing the gels were documented using a blue light transilluminator and a
camera to take a photo. If necessary, DNA bands of interest were excised from
the gel and the DNA was subsequently extracted using a QIAquick PCR
Purification Kit according to the manufacturer’s instructions.
Restriction, ligation and transformation
PCR products were digested with the restriction endonucleases HindIII and NdeI, and subsequently ligated using T4 DNA ligase into the
corresponding expression vectors pT7-7, which were also digested with the same
pair of restriction enzymes. The ligation mixture was transformed into
chemically competent E. coli DH5?
cells by the heat shock method to amplify the plasmid DNA and plated on Lysogeny
broth (LB) agar. Each step was performed using suitable buffer systems which
are listed with the other utilized components in Table A2.
PCR was used to verify successful cloning. Colony PCR was used as a fast method
to identify and screen for positive transformed clones containing the vector
with desired insert.
a standard colony PCR reaction a single bacterial colony was picked up from a
LB agar plate using a sterile toothpick and transferred into a PCR-tube
containing the PCR mix (Table A1). PCR was performed immediately and the
products were analyzed on an agarose gel with SYBR Green staining.
competent cells of BL21(DE3) E. coli
strain were transformed with the expression vectors (pT7-7::fdx4) extracted from DH5? for protein
production using standard methods.
overnight 5 mL preculture received from a single colony from an agar plate was
grown at 37 °C and 220 rpm in LB medium supplemented with 100 µg/mL
ampicillin. A 500 mL main culture was prepared as the starting culture
including the required ampicillin and additionally a pinch of
iron(II) sulfate was added and then inoculated by adding the preculture.
The cells were incubated at 37 °C in the shaker. When their OD600
reached 0.6, protein expression was induced by adding 0.1 mM isopropyl ??D?1?thiogalactopyranoside
(IPTG). The culture was left to grow at 37 °C until cell culture density
reached an OD600 of 5. Finally, the cells were harvested by centrifugation.
Cell lysis and protein purification
lysis was used as a method that disrupts cells and leads to the release of the
containing proteins into the lysis buffer. After cultivation, the bacteria cell
cultures were harvested by centrifugation (5,000 x g, 4 °C, 15 min) and the
cell pellet was resuspended in lysis buffer (Table A3) with an amount of three
times of the cell volume.
were lysed twice with the EmulsiFlex cell disruptor and the lysate was
centrifuged at 50,000 x g, 4 °C for 30 min to remove cell debris and high
molecular weight DNA.
resulting supernatant containing the soluble proteins was heated to 70 °C for
10 minutes, as this protein is from a hyperthermophilic organism and thus
denaturing most of the E. coli
proteins which were removed by another centrifugation step (15,000 x g, 20 °C,
supernatant was brought to 55 % saturation in ammonium sulfate, and the mixture
was stirred at 0 °C overnight. The precipitated proteins were again
centrifuged at 15,000 x g, 20 °C for 10 min and now the pellet was resuspended
in original volume of DEAE loading buffer (Table A3) for further purification
each centrifugation cycle a small amount of the supernatant and the pellet was
kept for further analysis by SDS-PAGE.
chromatography runs were performed using a Äkta Prime Plus (GE Healthcare) at
room temperature. For evaluation, absorbance at 280 nm and conductivity were
monitored and a chromatogram was recorded.
Ion exchange chromatography (IEX)
separates proteins due to their affinity of reversible adsorption to a counter
charged group immobilized on a matrix (DEAE Sepharose).
the column was appropriately equilibrated and A280 and the
conductivity baseline stabilized, AaFd4 diluted in lysis buffer was loaded onto
the column. Unbound material was washed with DEAE loading buffer (Table A3) and
subsequently the adsorpt protein sample was eluted with a flow rate of 2 mL/min
by gradually increasing the salt concentration with a linear gradient of DEAE
elution buffer (0 mM to 500 mM NaCl, Table A3). The eluates were collected in 2
mL fractions and analyzed by SDS-PAGE.