Objective of Dissertation - I-Shou University Institutional Repository:Item 987654321/18577

With enormous commercial values and environmental friendly advantage over traditional, chemically synthesized GABA and its analogues, studying GAD of diverse organisms had become interest of many researchers around the globe. GAD of P. abyssi has unusual ability to tolerate heat denaturation and still possesses catalytic activity to convert L-glutamate into GABA and CO2. Hence, the objective of this dissertation is to clone cDNA encoding a putative GAD into E. coli competent cell expression system, BL-21 (DE3).

To do so, an expression vector will be used to carry the cDNA. It is then transformed into BL-21 (DE3), and by adding suitable amount of inducer, the GAD will be synthesized rapidly and vastly. By modifying C-terminus of GAD with addition of poly-histidine, GAD can be easily purified and isolated from non-relevant enzymes and other cellular components. Purified GAD will be assayed based on its ability to catalyse formation of GABA as end product using proper amount of L-glutamate and PLP present in reaction buffer. Last but not least, GABA formed will be derivatized simply before analyzed and quantitated using HPLC equipped with UV/Vis detectors.

3. Materials & Method

3.1 Materials

Most materials, including organic solvents, mineral salts, E. coli culturing LB powder, antibiotics and buffers were purchased from Sigma-Aldrich® . Acetonitrile (HPLC grade) was purchased from Echo Chemical Co., Ltd. Expression vector, pET-51b(+) Ek/LIC vector kit was purchased from Merck Millipore. DNA polymerase and restriction enzymes were purchased from Promega. Dual color protein marker, TEMED, 30%

acrylamide/bis solution (29:1) for SDS-PAGE were purchased from Bio-Rad Laboratories, Inc.

3.1.1 1% Agarose Gel

1 g agarose powder was mixed with 100 ml ddH2O to obtain 1%

agarose gel for DNA gel electrophoresis [28]. Then, the mixture was heated until boiling using a microwave oven before casting into a mould with comb that leaves holes for DNA loading.

3.1.2 2.5% LB & LB Agar

To prepare 1 L LB (2.5%), 25 g LB powder was added to 1 L ddH2O.

Then, the mixture was autoclaved before inoculating bacterium. 15 g agar powder was added to 1 L ddH2O, together with 25 g LB powder for preparation of LB agar after autoclaving. Then, the mixture was placed into

Petri dishes. Ampicillin (100 mg/ml) was added to precooled LB/LB agar if necessary, and the final concentration should be 100 µg/ml. Avoid direct heat and UV irradiation onto ampicillin as they accelerate rapid degradation of antibiotics.

3.1.3 Binding Buffer for Cell Lysis

E. coli cells were collected after rPaGAD induction using 1 mM IPTG [29]. Then, the cells were re-dissolved in binding buffer, consisting 5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.4) before sonication for 10 mins at 80% amplitude, sonicate for 5 s and rest for 2 s interval. No EDTA was allowed in binding buffer as it strips nickel ions off Sepharose® affinity resin.

3.1.4 Nickel-Sepharose® Purification Buffers (pH7.4)

To charge Sepharose® beads, 50 mM NiSO4 was prepared (No specific pH). Wash buffer, 50 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl was prepared to elute non-relevant proteins from nickel-Sepharose® [30]

affinity column. Elution buffer with 500 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl was prepared to elute rPaGAD. Strip buffer containing 100 mM EDTA, 0.5 M NaCl, 20 mM Tris-HCl was meant to remove nickel ions from Sepharose® beads for storing.

3.1.5 SDS-PAGE & Western Blotting Preparation of SDS-PAGE

Solution components 12% resolving gel components

SDS-PAGE running buffer contained 25mM Tris, 192mM Glycine, and 0.1% SDS while Western blot transfer buffer was made by mixing methanol (99%) in SDS-PAGE running buffer. The final concentration of methanol was 10%. PBST contained 140mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4 and 5ml 10% Tween® 20. 2.5% blocking buffer for Western blotting contained 25g Skimmed milk powder dissolved in 50ml PBST. Coomassie Brilliant Blue R-250 used for SDS gel staining contained 0.75g R-250 powder dissolved in 5:5:1 ratio of

MeOH:ddH2O:acetic acid. 3X destained buffer for destaining SDS gel had 60% MeOH, 30% acetic acid, 10% ddH2O each. 4X SDS-gel loading buffer contained 2.5ml 1M Tris – HCl (pH 6.8), 0.5ml ddH2O, 1g SDS, 0.8ml 0.1%

Bromophenol Blue, 4ml 100% glycerol, 2ml 14.3M β-mercaptoethanol, adjusted to 10ml with ddH2O. SDS polyacrylamide gel intends to separate proteins based on their electrophoretic mobility. Samples would be boiled with anionic SDS loading buffer, linearizing and imparting evenly distributed negative charges onto polypeptides. Then, an electric current is applied across the gel and thus, samples travel towards anode. Larger proteins tend to travel slower than lighter ones due to larger retardation force exerts by the cross-linking of polyacrylamide molecules. 1° and 2°

antibodies were purchased from PerkinElmer. 1° antibody is immunoglobulin G that binds specifically towards polyhistidine and it is extracted from serum of lab mouse whereas 2° antibody is an anti-mouse immunoglobulin, coupling alkaline phosphatase. 2° antibody is meant to bind to the tail region of specific 1° antibody.

3.1.6 rPaGAD Catalytic Buffers

To investigate catalytic properties of rPaGAD, buffers consisting pH 3 to pH 10 were prepared. 50 mM sodium acetate buffers attributed pH 3 to pH 5 [31] whereas Good’s buffers attributed pH 6 to pH 10 [32]. pH value of each buffer was adjustable by either acetic acid towards acidity or NaOH towards basicity. Good’s buffer contained Bicine, CAPS, sodium acetate, Bis-Tris propane. Concentration of each chemical compound was 50 mM.

Reaction solution for rPaGAD catalytic conversion contained 50 mM L-glutamic acid and 2 mM PLP.

3.1.7 HPLC Eluent System

In the experiment, eluent system of HPLC utilized for GABA analysis consisted two mobile phases. Eluent A contained 3.54 g KH2PO4, 10.724 g Na2HPO4·7H2O and 5.66 g Na2HPO4 was prepared and adjusted to pH 7 with NaOH. Then, the final volume of eluent A was topped up to 1000 ml using ddH2O. 180 ml eluent A was mixed with 137.5 ml acetonitrile and 740 ml ddH2O. This was mobile phase/eluent A.

Mobile phase/eluent B was prepared by mixing 150 ml ddH2O and 850 ml acetonitrile. Both mobile phases were filtered with 0.22 µm filter unit, sonicated to remove air bubbles before practised in HPLC system.

3.1.8 GABA Derivatization Reagants

To derivatize GABA, borate buffer containing 3.1 g boric acid, 500 ml ddH2O, adjusted to pH 10 with NaOH was prepared. Then, 3 ml MeOH, 0.1 g OPA and 200 µl MPA were added to 40 ml borate buffer [94]. MeOH, OPA and MPA should be HPLC analytic grade (>99% purity).

3.2 Methods

3.2.1 rPaGAD cDNA Synthesis

Putative cDNA of rPaGAD (1173bp) synthesis was outsourced to PURIGO Biotechnology Co., Ltd. To maximize production of rPaGAD enzyme in E. coli competent cells (BL-21 DE3) expression system, codon optimization was employed on the cDNA that corresponded to E. coli codon usage [33]. rPaGAD cDNA was synthesized based on P. abyssi GAD protein sequence (GenBank: CAB50124.1) revealed on NCBI database (Appendix 8-9).

3.2.2 PCR of rPaGAD cDNA

Once cDNA of rPaGAD was synthesized, a pair of sense and antisense primers were designed for PCR, so that quantity of cDNA can be amplified exponentially. Both ends of rPaGAD cDNA would possess complementary extensions necessary to be cloned into expression vector after PCR using the primers. Besides, C-termini of rPaGAD would have polyhistidines (10X) inserted, so that these polyhistines are captured temporarily by Ni-Sepharose® beads during affinity purification. Primer synthesis was outsourced to Tri-I Biotech, Inc.

Sequence (5’  3’)

Forward Primer GAC GAC GAC AAG ATG AGC AAG TTC CCC GAA AAG GGT TTA CC

Reverse Primer GAG GAG AAG CCC GGT TAG TGG TGA TGG TGA TGG TGG TGA TGA TGC TTC AAA ACC TCC CTC AAG TC

Forward and reverse primers for PCR of rPaGAD

Forward and reverse primer sequences to amplify rPaGAD gene via PCR are shown above. Forward primer contains single enterokinase cleavage site before the start codon (highlighted in yellow box) that encodes methionine. In reverse primer, at least six histidines (not shown) are included at the end of rPaGAD gene before stop codon to ease purification of rPaGAD proteins via nickel affinity chromatography. Primers are synthesized by Tri-I Biotech, Inc. After PCR with this pair of primers, rPaGAD cDNA has complementary regions to that of pET-51b(+) Ek/Lic expression vector that ease the annealing without presence of DNA ligase.

To run 30 cycles of PCR, 23.5 µl 2X Taq Polymerase reaction buffer, 1 µl 10 mM sense/antisense primers each, 1 µl rPaGAD cDNA, 23.5 µl ddH2O were required, totaling 50 µl in volume.

3.2.3 T4 DNA Polymerase Treatment of rPaGAD cDNA

The last step before rPaGAD cDNA annealed to expression vector was treatment using T4 DNA Polymerase and dATP. It cleaved a portion of nucleotides at 3’ to 5’ direction of the cDNA [34], leaving overhang ends that were complementary to that of expression vector. 14.6 µl rPaGAD

cDNA, 2 µl 10X T4 DNA polymerase buffer, 2 µl dATP, 1 µl 100 mM DTT, 0.4 µl (2.5 U/µl) T4 DNA polymerase were required. T4 DNA polymerase was added at the last, making up 20 µl in total volume. The reaction started at 22°C for 30mins, and then shifted to 75°C for 20 mins. The treated cDNA was kept at -20°C for storage if necessary.

3.2.4 Annealing rPaGAD cDNA into pET-51b(+) Ek/LIC expression vector

Expression vector pET-51b(+) Ek/LIC (5218 bp) is ligation independent. Thus, no DNA ligase is required to anneal rPaGAD cDNA into this vector. Firstly, T4 DNA polymerase treated rPaGAD cDNA was incubated at 22°C for 5 mins. Then, 1 µl 25 mM EDTA was added to the cDNA, followed by adding 1µl pET-51b(+) Ek/LIC expression vector. Mix them thoroughly by stirring with a pipetman tip before incubating at 22°C for 5 mins. Then, the expression vector carrying desired gene was kept at -20°C for storage.

5’GACGACGACAAGATG

3’CTGCTGCTGTTCTAC rPaGAD cDNA (1173 bp) TGACCGGGCTTCTCCTC’3 ACTGGCCCGAAGAGGAG’5

T4 DNA polymerase + dATP

5’GACGACGACAAGATG

3’AC rPaGAD cDNA (1173 bp)

TGA’3

ACTGGCCCGAAGAGGAG’5

19 5’GACGACGACAAGATG

3’AC

rPaGAD cDNA (1173 bp) TGA’3

ACTGGCCCGAAGAGGAG’

+

5 GAT’5

CTACTGCTGCTGTTCT’3

5’CCGGGCTTCTCCTCA 3’T

pET-51 Ek/LIC vector 5218 bp

Annealing

3.2.5 Restriction Enzyme Digestion of rPaGAD/pET-51b(+) Ek/LIC for Physical Mapping

To ensure rPaGAD cDNA had been enclosed within pET-51b(+) Ek/LIC expression vector, initial checking was necessary before DNA sequencing. This can be done by physical mapping, running with restriction enzymes digested vector itself. Fortunately, the pET-51b(+) Ek/LIC vector has two restriction cutting sites specially engineered, that are AvrII and XbaI flanking rPaGAD cDNA (Appendix 8-8). Thus, 15 µl rPaGAD/pET-51b(+) Ek/LIC vector, 1.5 µl AvrII, 1.5 µl XbaI, 2 µl 10X restriction buffer and 5 µl ddH2O were mixed, placed at 37°C for 2 h. Then, 1 % agarose gel was casted. 5 µl Kb DNA ladder was loaded at one side of gel as gene sizing points. 5 µl restriction enzymes treated rPaGAD/plasmid sample was mixed

pET-51 Ek-LIC sequence

6391 bp

Av rII

rPaGAD

T7 promoter Strep Tag

XbaI

lac operator

with 1 µl loading dye before loading into gel as well. Then, the gel was run by 100 V for 20 mins.

To visualize DNA bands in agarose gel, the gel was immersed in solution containing diluted ethidium bromide (EtBr) for at least 15 mins.

Then, the gel was visualized under UV light along with camera installed at the top to capture fluorescing DNA bands. This is because UV irradiation caused intercalating EtBr to fluoresce orange color in the dark. Wearing protective gloves, lab coat and goggles is a must while handling EtBr because it is mutagenic.

3.2.6 DNA Sequencing of rPaGAD/pET-51b(+) Ek/LIC Expression Vector

To ensure nucleotide sequence of rPaGAD cDNA within pET-51b(+) Ek/LIC expression vector was correct, we employed DNA sequencing on the cDNA using third partner sequencing service, Tri-I Biotech, Inc. Sequencing primers chosen were T7 promoter and T7 terminator, which are sequences readily encoded in pET-51b(+) Ek/LIC expression vector. The result of DNA sequencing was aligned with cDNA of rPaGAD using Nucleotide BLAST to confirm there were no mutations (Appendix 8-13) before transformation into E. coli (BL-21 DE3) expression system.

3.2.7 DH-5α Transformation & Stock Preparation

To perform transformation of rPaGAD/pET-51b(+) Ek/LIC vector into E. coli DH-5α competent cells for storage purpose, 2 µl

rPaGAD/pET-22

51b(+) Ek/LIC vector was mixed with 20 µl DH-5α thoroughly and placed on ice for 30 mins, followed by instant transferring to the 42°C water bath added to each Eppendorf tubes before storing at -80°C as stocks.

3.2.8 rPaGAD/pET-51b(+) Ek/LIC Vector Extraction (Viogene Plasmid Extraction Kit)

A Viogene Plasmid Extraction Kit was purchased to ease the extraction of plasmid from DH-5α. To obtain fresh rPaGAD/pET-51b(+) Ek/LIC plasmid from DH-5α, tiny amount was scraped from stock of DH-5α and inoculated in 5 ml LB broth containing 5µl ampicillin (100 mg/ml). The broth was then cultured under 37 °C for 16 h. Centrifuged the broth with 4,000 G rotational speed for 15 mins to collect DH-5α pellet and decanted the supernatant. 200 µl MX1 buffer was loaded onto pellet to resuspend it completely, and vortex if necessary, followed by adding 250 µl MX2 buffer and gently mixed until clear solution was visible. The solution incubated at room temperature for 5 mins. 350 µl MX3 buffer was then added and mixed instantly until white precipitate formed. The mixture was centrifuged with 10,000 G for 10 mins, the supernatant was transferred into Mini Plus™

column with collection tube placed underneath. The column was centrifuged at 7000 G for 60 s, the flow through was discarded. Then, obtained 0.5 ml WN buffer to wash the column once, repeated 7000 G, 60 s centrifugation and wash with 0.7 ml WS buffer and repeated 7000 G, 60 s with all flow through discarded. The column was centrifuged at 10,000 G for 3 mins to remove residual ethanol, and then the collection tube was discarded and replaced with new 1.5 ml Eppendorf tube. 50 µl elution buffer was loaded onto membrane center of the column and allowed to stand for 3 mins. Lastly, the column was centrifuged at 10,000 G for 3 mins to collect plasmid that dissolved in elution buffer in Eppendorf tube. The elution buffer collected was reloaded onto membrane and centrifuged again to maximize amount of plasmids obtained, and these eluting steps had been repeated twice.

3.2.9 rPaGAD/pET-51b(+) Ek/LIC Plasmid Quantification

After extraction of rPaGAD/pET-51b(+) Ek/LIC plasmids from DH-5α, plasmid quantification was carried out using NanoDrop 2000C spectrophotometer. Firstly, 2 µl elution buffer was loaded onto nano-scaling platform of spectrophotometer and closed with the platform cover. Then, double clicked on “NanoDrop” software in computer, and chose Nucleic Acid option. Set the elution buffer as blank, then wiped it off with Kimwipes® tissue to ensure nothing was left behind. 2 µl plasmid was loaded at the same place and by clicking “Measure” the concentration of plasmid would be tabulated in ng/µl. Values of 260/280 and 260/230 ratios should fall around 1.8 and 2.0 respectively as these ratios indicate purity of DNA/RNA [35] extracted from cells.

3.2.10 Escherichia coli (BL-21 DE3) Transformation & IPTG Induction To transform rPaGAD/pET-51b(+) Ek/LIC plasmids into E. coli (BL-21) expression competent cells, 2 µl rPaGAD/pET-51b(+) Ek/LIC was mixed with 20 µl BL-21 and placed on ice for 30 mins before transferring instantly into 42 °C for 30 s. After heat-shocking, the BL-21 was placed on ice again, for 2 mins. 200 µl SOC/LB broth was added to the mixture and cuvette for spectrophotometry. With approximate 0.6~0.8 cell culture absorbance, BL-21 (DE3) was ready for rPaGAD induction.

1 ml (1 M) IPTG was added to 1 L LB broth containing BL-21 (DE3) to achieve 1 mM IPTG final concentration for induction at 16 °C for 16 h on 200 rpm shaker. After induction, entire LB broth was centrifuged and collected only pellet for sonication. The pellet was re-dissolved in binding buffer (Material 3.1.3) before sonication.

3.2.11 BL-21 (DE3) Competent Cell Lysis via Sonication

The sonicator probe was sprayed with 75 % EtOH, followed by ddH2O and dried with Kimwipes® tissue before sonication. Dissolved BL-21 (DE3) was placed on icy water and the probe was immersed into BL-BL-21 (DE3) solution. Amplitude of sonication was set at 80 %. Time interval

between sonication and rest were set at 2 s versus 4 s to prevent overheating of BL-21 (DE3) solution, and total sonication timeframe was 10 mins.

After sonication, the solution was centrifuged with 12,000 G rotational speed at 4 °C for 30 mins to isolate cell debris. Then, the supernatant obtained was filtered through 0.45 µm pore size filter unit to remove particles that would clog Ni-Sepharose® beads in column during affinity purification. The supernatant is kept in 4 °C fridge at all time before purification.

3.2.12 rPaGAD Purification via Ni-Sepharose® Affinity Column

Before affinity purification took place, 2 ml rPaGAD total protein solution was kept in 4 °C fridge. 2 ml Ni-Sepharose® beads was loaded into column tube, allowing them to settle underneath the tube before installing a piece of filter above the beads as buffering unit. Air bubbles were not allowed in the tube because they reduced affinity of the beads capturing polyhistidine-tag engineered at C-termini of rPaGAD [36].

3X beads volume of binding buffer (Material 3.1.3) were loaded into column with Ni-Sepharose beads to equilibrate environment of the beads, and the buffer flowed through without drying out the column. Then, rPaGAD total protein solution was flowed through the column as well and this step was repeated at least twice to maximize rPaGAD enzyme binding onto the beads. Elution of each flow-through solution was kept starting from this step onward. After that, 5X volume of wash buffer containing 50 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.4) was used to flush out non-relevant proteins from the column, and the elution of wash buffer was

kept as precaution measure. 2X volume of elution buffer containing 500 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.4) was used to elute rPaGAD from the column and the buffer was kept at 4 °C to preserve rPaGAD.

The column was then washed with 5X binding buffer, followed by 5X ddH2O and 3X 20 % EtOH before storing in 4 °C fridge for reusing purpose.

Ni-Sepharose® beads were soaked in 20 % EtOH to prevent growth of molds.

3.2.13 rPaGAD Buffer Exchange via Centrifugal Concentrator

Elution buffer (500 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.4) dissolved rPaGAD was exchanged to TE buffer because it is a better buffer in preserving protein. TE buffer contained 10 mM Tris and 1 mM EDTA, brought to pH 8 with HCl. Vivaspin® centrifugal concentrator was used to concentrate rPaGAD in elution buffer to a lesser volume via centrifugation. Then, small amount of TE buffer was added to concentrated rPaGAD solution, and centrifuged again to obtain adequate amount of TE buffer dissolved rPaGAD before further usage. All procedures were done either on ice or 4 °C to minimize heat denaturation of rPaGAD.

3.2.14 SDS-PAGE & Western Blotting of rPaGAD

SDS-PAGE and Western blotting were performed on rPaGAD to further confirm its identity [37]. For SDS polyacrylamide gel casting chemical proportion, please refer to section 3.1.5. Once SDS polyacrylamide

chemicals are mixed thoroughly, it is then poured into precast gel moulds and the gel would solidify within 15 mins. Stacking gel is casted after separating gel. Comb is placed on top of separating gel before casting stacking gel to provide hollow wells for sample loading.

60 µl rPaGAD total protein solution (original), 3X passed Ni-Sepharose® elution, wash buffer (50 mM imidazole) and rPaGAD elution (500 mM imidazole) each were mixed with 20 µl 4X SDS-gel loading buffer and boiled at 100 °C for 5 mins before loading into wells of SDS stacking gel. 25 µl boiled samples were loaded. 10 µl Prestained protein marker was loaded at the side of SDS gel as protein molecular weight reference in kDA unit. Two slices SDS-gels were required as one was to stain with Coomassie Brilliant Blue R-250 dye and another was to transfer onto PVDF membrane for Western blotting. Then, the gel units were run using 30 mA for 30 mins until the SDS-gel loading dye reached the bottom.

After that, a SDS-gel was stained for 15 mins on 50 rpm shaker, followed by destaining with 3X destain buffer until protein bands were visible. Another SDS-gel was sandwiched along with a 100 % methanol pre-treated PVDF membrane, two pieces filter papers at both sides, together with two pieces sponges covering both ends of filter papers to prevent desiccation of PVDF membrane and SDS-gel in the Western blotting cassette. Strictly no air bubbles were allowed between PVDF membrane and SDS-gel as protein transferring would be obstructed. Then, the cassette was immersed into transfer buffer-filled tank, with PVDF membrane facing anode of the

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