Title: Protein expression and purification

Objective: Large-scale production of soluble recombinant amyloid-?? peptide 1-42 using cold-inducible expression system
Amyloid-?? peptide 1-42 (A??1-42) plays major roles in the pathogenesis of Alzheimer’s disease, due to its aggregate nature, (A??1-42) has been difficult to manufacture in a soluble state in the bacterial expression system. In this study, expression system is altered to improve the soluble fraction of A??1-42 in Escherichia coli (E.coli) cells. The coding region of (A??1-42) was created in a pCold vector, pCold-TF to improve expression temperature. A??1-42 was expressed using pCold vector system recombinant mainly as a soluble protein and purified with a nickel-chalating resin. After studying the toxicity of the recombinant (A??1-42) on human neuroblastoma SH-SY5Y cells, the purified (A??1-42) induced cells toxicity on SH-SY5Y cells. In conclusion, this study delivers an important ways for the manufacture of aggregate prone-peptide such as (A??1-42)
The central events in the pathogenesis of the Alzheimer’s Disease (AD) are the aggregation and fibril formation of amyloid ‘?? peptides (A??). A?? the primary component of the amyloid plaque is composed of filamentous aggregates of 39-42 residue beta peptide produced by the proteolytic cleavage of amyloid precursor protein (APP). The abnormal fibrillar structures in senile disease are caused by the filament aggregates of A?? plaque which results in neurotoxicity. A??1-42 is a main component of amyloid plaques and it aggregation play a major role in the initiation of plaque and AD pathogenesis. Both in vivo and in vitro indicates A??1-42 aggregation and strong neurotoxicity. The nature of A??1-42 makes it challenging to produce in a soluble state for experimental investigation. There are many scientist that have been trying to express A??1-42 fused with Ubiquitin, glutathione-S-trans-ferase, or maltose binding protein, however the technique established for the protein manufacturing have numerous procedures or use toxic materials. The study involves the production of non-aggregated and soluble recombinant A??1-42 protein by optimizing the expression temperature and using pCold vector system. A??1-42 protein purified in this research induced cell toxicity or human neuroblastoma SH-SY5Y cells. The technique allows the efficient expression of soluble A??1-42, which can then be purified by an easy process.

‘ NI-NTA Agarose
‘ Mouse monoclonal anti ‘His
‘ Goat HRP- conjugated anti-mouse igG
‘ Westzol enhanced chemiluminescence
‘ (ECL) detection kit
‘ Thrombin
‘ Isopropyl-??-D-thiogalactopyranoside (IPTG)
‘ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
‘ Bromide (MTT)
‘ pCold-TF
‘ Commercial A??1-42
Expression of H6Ub- A??1-42
1. The E.coli BL21 (DE3) pLysS cells transformed the pET28a::H6Ub- A??1-42 were grown at 37?? C in 5ml of Luria-Bertani medium and induced with 0.5mM IPTG at 37 or 16?? C when absorbance reach 0.6 at 600nm.
2. Harvest and centrifuge the cells at 6000 rpm for 5 minutes at 4??C, after induction for 4hrs at 37??C or 12hrs at 16?? C.
3. Grow Induce cells without adding IPTG as control.
4. After centrifuge, re-suspend 22mg cells in 0.4ml lysis buffer and rupture by sonication for 15 s on ice.
5. After centrifugation at 12,000rpm for 15 mins, transfer supernatant to a new tube and the dissolved pellet in lysis buffer containing 8M urea.
6. Determine the protein concentration in the supernatant and dissolved pellet using Bradford method, bovine serum albumen (BSA) should be used as a protein concentration standard.

Analysis of western blot and quantification of protein yield
1. Separate the protein samples by 4’20% gradient SDS’PAGE and visualize by staining with Coomassie brilliant blue R250.
2. Measure the amount of H6Ub’Ab1’42 in the soluble and insoluble fractions,
3. Transfer the proteins on the gel electrophoretically to a nitrocellulose membrane, which is incubated with anti-His-tag and HRP-conjugated secondary antibodies.
4. Develop the blot using an ECL detection kit and analyse by using the
Bio-Rad gel documentation system. The levels of expression in the soluble and insoluble fractions were quantified by densitometer scanning of the film.

Expression and purification of Ab1’42 by pCold vector
1. Transfer the pCold’TF :: A??1-42 plasmid into E. coli BL21 (DE3) pLysS.
2. Grow the transferred cells at 37?? C in 1 L Luria’ Bertani medium and induced with 0.5 mM IPTG at 16?? C for 16 hrs.
3. Harvest the cells by centrifuging ( at 6,000 rpm, 10 min, 4??C)
4. Re-suspend 12 g (wet weight) cells in 50 mL lysis buffer.
5. After several cycles of freezing at -80??C and thawing at room temperature lyse the cells by sonication on ice, centrifuged (8,000 rpm, 30 min, 4??C), and the soluble fraction was collected.

Figure 1:
Schematic diagrams of recombinant Ab1’42 purification.
(A) The previously proposed method
1. The insoluble fraction is solubilized in 8 M urea buffer and then applied to a Ni’NTA column.
2. The eluate is buffer-exchanged into Tris buffer. Afterward, YUH-1 is added to the eluate for cleavage of the ubiquitin tag.
3. Further purification is performed by FPLC.
(B) The presently proposed method.
1. Applied soluble fraction to a Ni’NTA column.
2. Ab1’42 is cleaved and eluted in Tris buffer after washed
3. Additional purification can be done if needed
To purify TF-fused Ab1-42, filter supernatant using whatman filter paper. Allow the N-NA resin to settle in the column and equilibrate with 2 column buffer. Wash loaded cells lysis from the column with 5 column volumes of lysis buffer. The Ni’NTA-bound H6TF-fused Ab1’42 proteins were cleaved at 22??C for 8 hrs using 10 U (170 ??g) of thrombin in 10 mM Tris’HCl, pH 7.5, and processed Ab1’42 was eluted from the column. The purified Ab1’42 was dialyzed in 20 mM Tris, pH 7.5 and filtered with polyvinylidene fluoride (PVDF) syringe filter (pore size of 0.45 ??m). Then purified Ab1’42 was stored at -80??C until use. Throughout the freezing and thawing step, some aggregation occurs, so we used the supernatant after centrifugation (at 12,000 rpm, 15 min, and 4??C)

Cell toxicity assay
Human neuroblastoma SH-SY5Y cells (1 x 105) were seeded on 24-well plates at 37?? C and incubated for 12 hrs. Then, commercial and recombinant Ab1’42 was added at designated concentrations for 48 hrs. The cell viability was assessed using a MTT reduction assay. Briefly MTT was added to the cells at final concentration, 1mg/ml and incubated for 3hrs at 37??C. The 300 ??l of isopropanol was added to each well after media has been removed. By measuring the absorbance at 570 nm, MTT reduction was determined.


Figure 2:
The above figure denotes the improved efficiency of recombinant (A??1-42) engenderment by optimising the expression temperature (A). E. coli BL21 (DE3) pLysS, transformed with H6Ub’Ab1’42, was collected before (-) and after (+) IPTG induction at 37 or 16??C. Soluble and insoluble proteins were disunited by 4-20% SDS’PAGE and stained with Coomassie blue. The proteins expressed were indicated by arrowhead. (B) Soluble (S) and insoluble (P) H6Ub’ Ab1’42 induced at 37 or 16??C was divided by SDS’PAGE and exposed to immune blotting with an anti-His antibody (upper panel). Protein expression at 37 or 16??C was measured by densitometric scanning of the immunoblot shown above. The strengths of the S and P proteins expressed at 16??C were compared with those of the proteins expressed at 37??C, the expression levels of which were set at 100%.

Figure 3:
The above diagram indicates the expression and purification of recombinant Ab1’42 utilizing pCold vector. (A) .The Ab1’42 gene was inserted into pCold’TF containing a hexahistidine sequence to construct the pCold’TF::Ab1’42 expression plasmid. (B) Ab1’42 was expressed in E. coli BL21 (DE3) cells transformed with pCold’TF::Ab1’42. The H6TF-fused Ab1’42 protein obtained using Ni’NTA-agarose column was sliced by thrombin, and mature Ab1’42 was purified. The soluble proteins from E. coli changed with pCold vector (lane 1) or pCold-TF::Ab1’42 (lane 2) and purified Ab1’42 (lane 3) were divided by 4’20% gradient SDS-PAGE and visualized with Coomassie blue.

Process for recombinant Ab1’42 purification
Irrespective of importance of Ab1’42, the poor solubility of Ab1’42 makes it challenging to examine its function and pathological effects. Currently, chemically synthesized Ab1’42 is mainly utilized for experiments, however it fundamentals to be solubilized in a dimethyl sulfoxide-containing buffer by sonication, and it is costly. To address this, we developed a method by which Ab1’42 was engendered with an ubiquitin extension. In this technique, the insoluble ubiquitin’Ab1’42 fusion protein was solubilized in 8 M urea buffer and purified by affinity chromatography.
Subsequently, Ab1’42 was cleaved from the fusion protein utilizing yeast ubiquitin hydrolase-1 (YUH-1) and purified by reverse-phase chromatography. Even though Ab1’42 can bring about relatively enormous amounts in this way, this process have multiple steps, as well as solubilizing the aggregated fusion protein in denaturing urea buffer. In this discussion we report an improved ways that increases the soluble fraction by utilizing a pCold vector system and an optimized expression temperature. Since the soluble fraction is directly applied to Ni’NTA column, it is not indispensable to solubilize the aggregated Ab1’42 pellet. Additionally, utilization of the pCold’TF vector further simplifies downstream purification processes, including cleavage and elution. This tactic may be benefits to boost the solubility and yield of the protein of interest.
The study shows that the toxic effect of recombinant Ab1’42 purified from cold inducible system was studied on human neuroblastoma SH-SY5Ycells. Cells were incubated with Ab1’42 at indicated concentrations for 48 hours; viability of the cells was analysed using MTT reduction assay. Purified Ab1’42 did not affect the cytotoxicity. The cell viability of Ab1’42 on SH-SY5Y cells decreased in a concentration. These indicate that the recombinant Ab1’42 purified using pCold’TF vector and cold-inducible condition in this study had the instinctive nature of Ab1’42 toxicity which can affect cell viability. Cold-inducible purification method to purify Ab1’42 has fewer and easier purification steps than our previous ubiquitin fusion technique. Nevertheless recombinant Ab1’42 purified in this study had slightly but significantly more active cytotoxicity than commercial and recombinant Ab1’42 purified from ubiquitin fusion method. Thus, we propose that our technique will be beneficial to get a large quantity and high quality of aggregation-prone peptides such as Ab1’42 .

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