Themethod of PCR was used for the rapid and specific amplification ofDNA-fragments of interest, here pUC18 vectors with the fdx4-insert. Besidesrestriction sites within the PCR-primer sequences were built in for latercloning steps, too. The standard PCR protocol and the components for the PCRreaction which were used, are shown in Table A1. Annealing temperature andelongation time were adjusted based on primer characteristics and the productsize, respectively. PCR products were analyzed and separated by agarose gelelectrophoresis.
These separated DNA fragments were excised and purified usinga PCR purification kit. 1.1 Agarose gel electrophoresisAgarosegel electrophoresis was performed to separate DNA fragments according to theirsize and therefore used to confirm each step of the cloning process. Agarosegel was prepared by adding appropriate amount of agarose into TAE buffer,samples were mixed with 6x loading dye including SYBR Green and the DNAfragments were separated in an electric field (80-120 V).Theagarose concentration of the gels was set to 1.
0 % according to the size of DNAfragments to be analyzed. A 1 kbp DNA marker was applied as a size standard.Uponfinishing the gels were documented using a blue light transilluminator and acamera to take a photo. If necessary, DNA bands of interest were excised fromthe gel and the DNA was subsequently extracted using a QIAquick PCRPurification Kit according to the manufacturer’s instructions. 1.2 Restriction, ligation and transformationPurifiedPCR products were digested with the restriction endonucleases HindIII and NdeI, and subsequently ligated using T4 DNA ligase into thecorresponding expression vectors pT7-7, which were also digested with the samepair of restriction enzymes. The ligation mixture was transformed intochemically competent E. coli DH5?cells by the heat shock method to amplify the plasmid DNA and plated on Lysogenybroth (LB) agar.
Each step was performed using suitable buffer systems whichare listed with the other utilized components in Table A2. 1.3 Colony PCRAdditionally,PCR was used to verify successful cloning. Colony PCR was used as a fast methodto identify and screen for positive transformed clones containing the vectorwith desired insert.Fora standard colony PCR reaction a single bacterial colony was picked up from aLB agar plate using a sterile toothpick and transferred into a PCR-tubecontaining the PCR mix (Table A1). PCR was performed immediately and theproducts were analyzed on an agarose gel with SYBR Green staining.
1.4 Protein expressionChemicallycompetent cells of BL21(DE3) E. colistrain were transformed with the expression vectors (pT7-7::fdx4) extracted from DH5? for proteinproduction using standard methods.Anovernight 5 mL preculture received from a single colony from an agar plate wasgrown at 37 °C and 220 rpm in LB medium supplemented with 100 µg/mLampicillin. A 500 mL main culture was prepared as the starting cultureincluding the required ampicillin and additionally a pinch ofiron(II) sulfate was added and then inoculated by adding the preculture.
The cells were incubated at 37 °C in the shaker. When their OD600reached 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 densityreached an OD600 of 5. Finally, the cells were harvested by centrifugation. 1.5 Cell lysis and protein purificationCelllysis was used as a method that disrupts cells and leads to the release of thecontaining proteins into the lysis buffer. After cultivation, the bacteria cellcultures were harvested by centrifugation (5,000 x g, 4 °C, 15 min) and thecell pellet was resuspended in lysis buffer (Table A3) with an amount of threetimes of the cell volume.Cellswere lysed twice with the EmulsiFlex cell disruptor and the lysate wascentrifuged at 50,000 x g, 4 °C for 30 min to remove cell debris and highmolecular weight DNA.
Theresulting supernatant containing the soluble proteins was heated to 70 °C for10 minutes, as this protein is from a hyperthermophilic organism and thusdenaturing most of the E. coliproteins which were removed by another centrifugation step (15,000 x g, 20 °C,10 min).Thesupernatant was brought to 55 % saturation in ammonium sulfate, and the mixturewas stirred at 0 °C overnight. The precipitated proteins were againcentrifuged at 15,000 x g, 20 °C for 10 min and now the pellet was resuspendedin original volume of DEAE loading buffer (Table A3) for further purificationby chromatography.Aftereach centrifugation cycle a small amount of the supernatant and the pellet waskept for further analysis by SDS-PAGE. 1.6 Chromatographic techniquesAllchromatography runs were performed using a Äkta Prime Plus (GE Healthcare) atroom temperature.
For evaluation, absorbance at 280 nm and conductivity weremonitored and a chromatogram was recorded. 1.6.1 Ion exchange chromatography (IEX)IEXseparates proteins due to their affinity of reversible adsorption to a countercharged group immobilized on a matrix (DEAE Sepharose).Afterthe column was appropriately equilibrated and A280 and theconductivity baseline stabilized, AaFd4 diluted in lysis buffer was loaded ontothe column. Unbound material was washed with DEAE loading buffer (Table A3) andsubsequently the adsorpt protein sample was eluted with a flow rate of 2 mL/minby gradually increasing the salt concentration with a linear gradient of DEAEelution buffer (0 mM to 500 mM NaCl, Table A3).
The eluates were collected in 2mL fractions and analyzed by SDS-PAGE.