2.7.1 Synthetic DNA
Synthetic DNA containing the full length human POPDC1 sequence was purchased from Genewiz (US). The fragment was codon optimised for expression in E. coli. Synthetic POPDC1 gene:
5’ATGAATTACACCGAAAGTAGCCCGCTGCGTGAAAGCACCGCAATCGGCTTCACGCCCG AACTGGAAAGCATTATCCCGGTGCCGAGCAATAAGACCACTTGTGAGAATTGGCGCGAA ATCCACCATCTGGTTTTCCATGTTGCAAACATTTGCTTCGCCGTTGGTTTAGTTATTCCT ACCACTTTACATTTACACATGATCTTTCTGCGCGGTATGCTGACTTTAGGTTGCACTTTA TATATCGTTTGGGCCACTTTATATCGCTGCGCTTTAGATATCATGATTTGGAATAGCGTG TTTTTAGGTGTGAACATTTTACATTTAAGCTATTTACTGTATAAGAAGCGTCCCGTTAAG ATTGAAAAGGAGCTGAGCGGCATGTACCGTCGTTTATTCGAACCTCTGCGCGTGCCTCC CGATCTGTTTCGCCGTCTGACTGGTCAATTCTGCATGATCCAGACTTTAAAGAAGGGCCA AACCTATGCCGCCGAAGACAAAACCAGCGTTGATGACCGTTTAAGCATTTTACTGAAGGG
CAAAATGAAGGTGAGCTATCGTGGTCATTTTTTACACAACATTTATCCGTGCGCCTTCAT CGATAGCCCGGAATTCCGTAGTACCCAGATGCACAAAGGCGAGAAGTTCCAAGTTACCA TCATCGCCGACGACAATTGTCGTTTTTTATGCTGGAGCCGCGAGCGTCTGACCTATTTTC TGGAGAGCGAGCCGTTTCTGTACGAGATCTTTCGCTATTTAATCGGTAAAGATATTACCA ATAAGCTGTATTCTTTAAACGACCCGACTTTAAATGACAAAAAGGCCAAAAAATTAGAAC ACCAGCTGTCTTTATGCACCCAGATTAGCATGCTGGAGATGCGCAACAGCATCGCCAGC AGCAGTGATAGCGATGACGGTTTACATCAGTTTCTGCGTGGCACCAGCAGCATGAGTTC TTTACATGTTAGCAGTCCGCATCAGCGTGCCAGCGCAAAAATGAAGCCGATTGAGGAAG GTGCTGAAGATGATGACGACGTGTTTGAACCGGCAAGCCCGAACACTTTAAAAGTTCAT CAGCTGCCG 3’
2.7.2 Molecular cloning
Fragments of POPDC1 cDNA were created and amplified by PCR using a set of primers designed to bind the protein at various domains and all reactions were carried out in a MJ Research PTC-200 Thermal Cycle (MJ Research, Nevada, USA) (Table2). PCR reaction conditions were constant throughout all experiments with only the annealing temperatures changed depending on primers used. The PCR machine conditions were as follows: 95oC for 2 mins, then 30 cycles of 95oC
for 30 seconds, primer specific annealing temperature (Table 2) for 30 seconds, 72oC for 30 seconds, followed by 72oC for 5 minutes then 4oC on hold. Sequences
5’TAC TTC CAA TCC and 5’TAT CCA CCT TTA were added to the 5’ and 3’ primers respectively to allow for ligation independent cloning into the pNIC28-Bsa4 plasmid vector (appendix 1) (Keates et al., 2011). NEBuilder® HiFi DNA Assembly Cloning Kit was used following the manufacture’s protocol to insert the created fragments into the plasmid vector, which was linearized by BsaI digest. To ensure correct sequences were produced, fragments were initially transferred into DH5α competent cells (NEB) using manufacturers protocol. Positive colonies were subjected to colony PCR to confirm presence of transformed fragments prior to being transformed into BL21 (DE3) competent cells (NEB). A single colony was taken, resuspended in 10 µl dH2O and added to a PCR reaction
including; 10 mM 5’ primer (Table 2), 10 mM 3’ primer (Table 2), and taq polymerase master mix. Examination of the fragments by agarose gel
electrophoresis identified successful transformations. Using Monarch® DNA Gel Extraction Kit (New England Biolabs, US), confirmed fragments were excised and extracted from the gel for transformation into BL21 (DE3) competent cells.
Successful BL21 (DE3) colonies were miniprepped and sequenced (Source Bioscience, UK; Eurofins Scientific, UK). From confirmed transformation reactions, single colonies were picked and grown in LB supplemented with 50 mg/mL kanamycin to allow for glycerol stocks to be produced. 1 mL of culture was added to 1 mL of sterilised 50 % glycerol and stored at -80 oC.
Table 2: Primers designed for synthetic POPDC1 fragment generation
Name Sequence Tm (OC)
Full length 5’ TAC TTC CAA TCC ATG AAT TAC ACC GAA 52
Popeye domain 5’ TAC TTC CAA TCC AAG CGT CCC GTT AAG 55
Full length 3’ TAT CCA CCT TTA CGG CAG CTG ATG AAC 55
Popeye domain 3’ TAT CCA CCT TTA CTT AGG CCT TTT TGT C 52
2.7.3 Solubility testing
Expression screening was carried out to determine optimal solubility conditions for large scale purification. Scrapings from glycerol stocks were grown in 10 mL LB supplemented with 50 mg/mL of kanamycin overnight at 37oC whilst shaking
at 220-225 rpm. The following day, three 50 mL Conical Centrifuge Tubes
(Corning, UK) with 10 mL of fresh LB were inoculated with 1 mL of the overnight culture and grown to OD600 0.6 - 0.8 whilst shaking at 220-225 rpm. Prior to the
induction of protein expression, 1 mL of the culture was removed and added to a fresh 1.5 mL Eppendorf. Samples were centrifuged at 14,000 x g for 10 minutes; the supernatant was discarded, and the pellet was stored at -20o C. Protein
expression was induced by the addition of one of three IPTG concentrations; 0.1 mM, 0.4 mM or 1 mM. Cultures were allowed to express for 3 hours at 37oC or 16
hours at 16oC whilst shaking at 220-225 rpm. At the end of the induction period
an OD600 reading was taken to allow the samples for analysis via SDS-PAGE to be
same manner as the pre-induction sample. Prior to SDS-PAGE, samples were defrosted and 1 mL of Bugbuster® (Merck) was prepared and supplemented with 25 units of Benzonase (Sigma-Aldrich). 60 µL of Bugbuster® plus Benzonase was added to each pellet and pipette thoroughly to resuspend all cells. Samples were incubated at room temperature for 20 minutes to allow for lysis of the cell
membrane. Each sample was made up to a final volume (µL) of 150 x OD600 with
water. To a fresh Eppendorf, 5 µL was removed from each pre-induction and post-induction sample and a further 10 µL of nuclease-free water was added. These samples represent the total protein in both pre-induction and post-
induction cultures. Eppendorf tubes were centrifuged at 14,000 x g for 5 minutes and the supernatant from each condition was transferred to a fresh 1.5 mL Eppendorf and 15 µL was transferred to a further labelled tube, this represents the soluble fraction. The pellet was resuspended in the original volume of water. 5 µL was transferred to a fresh Eppendorf and a further 10 µL of water was added, this sample represents the insoluble fraction. Immediately prior to running the gel, 5x Laemmli protein sample buffer (Bio-Rad) was added and tubes were heated at 60oC for 10 minutes. SDS-PAGE analysis was carried out as
described in 2.8.1.
Each construct was subjected to all test conditions.
2.7.4 Large scale protein purification
After identification of optimal growth conditions in the solubility screening tests, large scale expression was carried out following the same protocol as described in 2.6.1 and 2.6.2).
Cultures were pelleted by centrifugation at 6000 x g for 15 minutes. Cell pellets were briefly frozen at -80oC, thawed and the pellets were resuspended in lysis
buffer (20 mM Tris-HCl; 0.5 M NaCl, pH7.4). Lysate was transferred to a 50 mL Conical Centrifuge Tubes (Corning, UK) and samples were sonicated on ice for 30 minutes, with 15 seconds of sonication and 15 seconds on ice. Cell debris were pelleted by centrifugation at 16,000 x g for 45 minutes. During this time, 1 mL of Ni-NTA resin (Qiagen) was allowed to settle in disposable Econo-Pac®
Chromatography Column (Bio-Rad) and washed thoroughly with water then equilibrated with binding buffer (20 mM Tris-HCl; 0.5 M NaCl; 5 mM Imidazole,
pH7.4). Two of these columns were stacked allowing for lysate to flow through Ni-NTA resin twice capturing as much of the expressed protein as possible. After centrifugation, the supernatant was added to columns and allowed to drip through. Flow through lysate was collected and a 15 µL sample was taken for quality control. The columns were separated and resin bed was washed five times with binding buffer (20 mM Tris-HCl; 0.5 M NaCl; 5 mM Imidazole, pH7.4) and a further three times with wash buffer (20 mM Tris-HCl; 0.5M NaCl; 20 mM Imidazole, pH7.4). Protein elutions were collected in 500 µL of elution buffer (20 mM Tris-HCl; 0.5M NaCl; 0.3 M Imidazole, pH7.4). 15 µL of each elution was taken for analysis on SDS-PAGE following protocol outlined in section 2.8.1. Immediately prior to running, 5x Laemmli protein sample buffer (Bio-Rad) was added and samples were heated to 60oC.