Prokaryotic Expression, Purification and Identification of Human PDE9A Protein

Cerebral aggregation of beta amyloid plaques (Aβ) and neurofibrillary tangles is responsible for the onset of Alzheimer’s disease (AD), and PDE9A inhibition rescues Aβ-induced deficits in synaptic plasticity and cognition. This study was aimed to express active PDE9A protein for subsequent inhibitor screening. The PDE9A gene was cloned from human cDNA by real-time polymerase chain reaction, and then the gene sequence and its amino acid sequence were analyzed on Lasergene. An inducible expression vector was constructed by enzyme digestion-seamless cloning and transformed into Escherichia coli BL21 (DE3) for PDE9A expression with isopropyl β-D-1-thiogalactopyranoside (IPTG) as an inducer. The recombinant protein was purified by Ni-NTA affinity chromatography and its activity was determined by a phosphodiesterase assay kit. It was found the open reading frame of PED9A was 1035 bp long, the deduced protein was composed of 345 amino acids, and its predicted isoelectric point was about 4.84. The E. coli vector ST6-PDE9A successfully expressed the recombinant PDE9A protein in the supernatant of bacterial lysate. The optimal culture conditions were that the bacterium ST6-PDE9A was grown first in a lysogeny broth at 37 oC to an OD600 of 0.6-0.8 and then at 16 oC for 40 h with the addition of 1 M IPTG. Activity test showed PDE9A significantly hydrolyzed the substrate cyclic guanosine monophosphate. In conclusion, we constructed a prokaryotic expression vector and expressed active proteins, laying a solid foundation for screening PDE9 inhibitors. Journal of Biology and Life Science ISSN 2157-6076 2019, Vol. 10, No. 1 23


Introduction
Alzheimer's disease (AD) is a progressive, unremitting and neurodegenerative disorder that affects wide areas of the cerebral cortex and hippocampus (Masters et al., 2015). In AD patients, memory loss is accompanied by the formation of beta-amyloid (Aβ) plaques and the appearance of pathological tau (Garcia-Osta, Cuadrado-Tejedor, Garcia-Barroso, Oyarzabal, & Franco, 2012). An estimated 24 million people worldwide have dementia, and the majority are thought to have AD (Ballard et al., 2011). The increasing number of affected individuals, the long duration of disability, and the rising costs of care for AD have posed heavy burdens to our society (Sibener et al., 2014). However, the etiology and pathogenesis of AD are still poorly understood. Evidence shows seven potential modifiable factors (diabetes, midlife hypertension, midlife obesity, physical inactivity, depression, smoking, and low educational attainment) contribute to the risk of AD (Norton, Matthews, Barnes, Yaffe, & Brayne, 2014). Recent genome-wide association research has identified a number of risky genes that influence the susceptibility to late-onset AD (Jiang, Yu, Tian, & Tan, 2013).
Phosphodiesterase (PDE) 9 is the most affinitive to cyclic guanosine monophosphate (cGMP) in the PDE family (Wang, Wu, Egan, & Billah, 2003), which is distributed widely in the central nervous system. Aging is associated with an increase in PDE9 expression and activity and a decrease in cGMP concentration (Domek-Lopacinska & Strosznajder, 2010). PDE9A inhibition can rescue Aβ-induced deficits in synaptic plasticity and cognition (Kroker et al., 2014). The PDE9A inhibitor BAY 73-6691 can enhance early and late long-term potentiation (Lopez et al., 2012). Another PDE9A inhibitor PF-04447943 exhibits precognitive activity in several rodent models and synaptic stabilization in an amyloid precursor protein transgenic mouse model (Verhoest et al., 2012). However, a phase-2 study demonstrates a 12-week PF-04447943 treatment did not improve cognition, behavior or global change compared with the placebo (Schwam et al., 2014). New PDE9A inhibitors are unwarranted to be potential drugs for AD treatment.
In this study, the PDE9A gene was cloned from the human body and a prokaryotic expression vector was constructed. Then PDE9A recombinant protein in Escherichia coli BL21(DE3) was expressed and then purified by Ni+ column affinity chromatography. PDE9A activity was detected by a PDE-Glo TM phosphodiesterase assay kit.

Primer Design
The opening reading frame of the PDE9A gene was obtained from Pubmed. The nucleotide sequence corresponding to the 241-566 amino acid of subtype PDE9A was selected as the target to design a pair of primers. The following upstream and downstream primers were added to the SFI I restriction site:

Expression Vector Construction
The target gene fragment and ST6 plasmid after SFI digestion were put together for seamless cloning in a 10 μL system, which contained master assembly buffer mix (5 μL), ST6 vector after SFI digestion (2.5 μL), and the gel fraction (2.5 μL). After complete mixing, the reaction system was placed in a water bath at 50 º C for 30 min. Then the seamless cloned product was transformed into E. coli DH5α, which was plated on AMP (100 mg/mL)-containing plates and cultured overnight at 37 º C. Positive clones were selected by colony PCR and sent to Taihe Biotechnology Co., Ltd for sequencing.

Induced Expression and Determination of Recombinant Protein
The successfully sequenced colonies were shaken overnight at 37 º C. Plasmid PDE9A-ST6 was extracted with an Aidlab plasmid rapid extraction kit (Cat. No. PL02), converted into the E. coli BL21(DE3) strain, and frozen at -80 ºC. After that, 50 μL of the frozen bacterial solution was placed in 5 mL of a terrific broth (LB) medium and activated overnight at 37 º C for 14 h. Then it was transferred to 100 mL of a fresh medium and cultured at 37 º C until the OD 600 reached 0.6-0.8. After that, 100 μL of 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) was added for 40 h of induction at 16 º C. The cells were collected, sonicated and then centrifuged at 12,000 rpm for 30 min. Then 10 μL of each denatured sample (whole protein, supernatant, and precipitate) was taken for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by CBB-R250 staining.

Purification and Activity Detection of Recombinant Protein
The supernatant of the PDE9A-expressing strain was purified using Ni + column affinity chromatography (equilibration buffer: 10 mM imidazole; washing buffers: 20 and 50 mM imidazole; elution buffer: 250 mM imidazole) and gel chromatography. Then the eluted recombinant protein was dialyzed against a solution containing no salt or imidazole (volume ratio: 1:100, liquid changed every 12 h, dialysis for 36 h). The purified protein was quantitatively analyzed by the bicinchoninic acid (BCA) method. The PDE9A activity was assayed using a PDE-Glo TM PDE assay kit (Promega) according to the manufacturer's instructions. The PDE9A activity was presented as the relative hydrolysis rate and normalized to the assayed proteins.

Amplification of the Target Fragment
The open reading frame of PED9A was 1035 bp long, and the deduced protein was composed of 345 amino acids; its predicted isoelectric point was about 4.84. The target gene fragment was amplified by PCR and then detected by 1% agarose gel electrophoresis. Our results indicate the target band size is correct (Figure 1). The target protein was cleaved for gel recovery to construct a recombinant vector.

Restriction Enzyme Digestion of Recombinant Vector
The correctly-sequenced plasmid was subjected to restriction enzyme verification. Two bands appeared after digestion, and the electrophoresis position was consistent with the sizes of the ST6 vector (6402 bp) and the target fragment (1035 bp), indicating the prokaryotic expression vector of ST6-PDE9A was successfully constructed (Figure 2).

Soluble Analysis of Recombinant Protein PDE9A
The cells after expression induction were collected by centrifugation and resuspended in phosphate buffer solution (PBS). After ultrasonication, the supernatant and precipitate were collected for SDS-PAGE. It was found the supernatant and precipitate showed clear bands around 39 KD, which was consistent with the expected band size, but the precipitate was more obvious. This result indicates the target protein mainly existed as inclusion bodies ( Figure 3).

Protein Purification and Expression Condition Optimization
After the Ni + column affinity chromatography, SDS-PAGE showed the recombinant protein eluted with 250 mM imidazole was about 39 KD in size, with clear and few bands (Figure 4). We explored the expression conditions of the transformed positive strains from multiple aspects (media, temperature, induction time, IPTG concentration). The expression induction media were tryptone yeast extract (YT), terrific broth (TB) and lysogeny broth (LB). The expression levels in YT were significantly higher than in TB and LB ( Figure 5). The other best conditions were 16 º C, 40 h and 1 M IPTG.

Protein Activity Test
The concentration of the recombinant protein after dialysis was 0.8046 mg/mL. Then the the purified protein was diluted by 2,4,8,16,32,64,128 and 256 fold. The substrate cGMP, termination buffer, detection buffer and Kinase-Glo○ R reagent were separately added. The luminescence after the complete reaction was detected on a microplate reader (Spark, Tecan, Switzerland). It was found the PDE9A protein at different concentrations could all hydrolyze the substrate cGMP. The substrate hydrolysis was accelerated with the increasing protein concentration. The EC 50 was 0.08347 mg/mL identified by fitting the curve to GraphPad.

Discussion
The exponential rise in the number of AD cases imposes heavy emotional and financial burdens onto families and communities. So far, acetylcholinesterase inhibitors and memantine are the only drugs approved for AD treatment (Anand, Gill, & Mahdi, 2014). These drugs only improve symptoms, but less affect the disease process (Anand et al., 2014). Insufficient understanding about the pathogenic process may be the main reason for the non-availability of effective treatment (Kumar, Singh, & Ekavali, 2015). The cAMP and cGMP signalings are both involved in various cellular functions, including neuroplasticity and neuroprotection (Heckman, Wouters, & Prickaerts, 2015). PDE inhibitors enhance cAMP and/or cGMP signaling via reducing the degradation of these cyclic nucleotides (Heckman, Blokland, Ramaekers, & Prickaerts, 2015). PDE inhibitors have been considered to be therapeutic targets for AD. ISSN 2157-6076 2019 PDE9, a representative member of the PDE family, hydrolyzes cGMP and has many subtypes, such as PDE9A1, PDE9A2 and PDE9A3. Two PDE9A inhibitors (BAY 73-6691 and PF-04447943) reportedly enhance synaptic plasticity and cognitive function in rodents (Hutson et al., 2011;van der Staay et al., 2008). The PDE9 enzyme preparation and screening methods are quite different between these two inhibitors. For BAY 73-6691, PDE9A was recombinantly expressed in Sf9 insect cells using a pFAST-BAC baculovirus expression system (Invtrogen, CarIsbad, CA) (Wunder et al., 2005). The [ 3 H] cAMP and [ 3 H] cGMP scintillation proximity assays (GE healthcare, Buckinghamshire, UK) were used for enzyme inhibition research (Wunder et al., 2005). For PF-04447943, the rhesus PDE9A2 construct (GenBank ID# FJ914626) was subcloned into a pcDNA3.3 TOPO vector (Invitrogen) and HEK 293 cells (Hutson et al., 2011). The PDE9A inhibitors were screened using a cGMP CL HitHunter assay kit (Hutson et al., 2011). The recombinant protein expressed in vitro by E. coli is an important tool in biotechnology research owing to the simple operation, low cost, high expression efficiency, stable product and easy identification. In this study, we selected the E. coli BL21(DE3) strain as a recipient bacterium, which was different from previous studies. In addition, [ 3 H] cAMP or cGMP causes less environmental pollution and is unsuitable for use all the time. Our test kit for PDE9 activity is featured by low cost, high quality and little environmental pollution.

Journal of Biology and Life Science
We also explored the conditions for inducing the expression of recombinant PDE9A protein.
When the expression temperature is too high, the bacteria grow too fast and the target protein cannot be folded correctly to form more inclusion bodies, but the cell growth is slow at too low temperature. Thus, suitable temperature is conducive to protein expression. As reported, the bacterium (pET32a-PDE2) was grown first in LB at 37 º C to an OD 600 of 0.6-0.8 and then at 16 º C for 6 h by adding 0.5 mM IPTG (Zhu et al., 2013). On this basis, we optimized the protein expression conditions to be: YT, 16 º C, 40 h, and 1 mM IPTG.
In conclusion, we successfully constructed a PDE9A-ST6 expression vector and expressed PDE9A at 16 º C for 40 h. Based on the optimal conditions, the purified PDE9A was obtained by Ni+ column affinity chromatography. This study underlies future preparation and functional study of PDE9A protein inhibitors.