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pHMT-LbCas12a plasmid (LbCas12a protein purification)

To express and purify Cas12a protein, we combine T7 promoter, poly Histidine-tag (10XHis), Maltose binding protein (MBP), and TEV recognizing motif with Cas12 coding sequences. T7 promoter can produce high yields of protein in E. coli with T7 RNA polymerase expression. 10XHis and MBP are used for protein purification through their high affinity to Nickel (II) ion and maltose, respectively. TEV recognizing motif separate 10XHIS and MBP from Cas12a, which is used to elute untagged Cas12a protein after purification. The composition 10X His-MBP-TEV site-Cas12a is called pHMT-Cas12a.

Experiment Results

• Introduction

In our project, we combined three parts of biological reactions to detect ASFV specific sequence in samples. The first one is LbCas12a-crRNA system, which can specifically recognize ASFV specific double stranded DNA (dsDNA) sequence on P72 gene. The secondary part is the trans-activation of LbCas12a-crRNA system. When LbCas12a-crRNA system binds to ASFV specific dsDNA sequences, LbCas12a-crRNA system will cleave dsDNA and further degrade non-specific single stranded DNA (ssDNA). To detect the degradation of ssDNA in ASFV-activated LbCas12a-crRNA system, we will use the PicoGreen fluoresce dye to monitor the undegraded ssDNA, which is the third part. To transfer reaction from part I/II/III to detection, we plane to conjugate ssDNA on magnetic beads. The ssDNA conjugated magnetic beads will be easily captured and transfer by electromagnetic force. In the following result section, we will show our progress through experiments that supported our project design.

• Our targets

Steps to establish CRISPR-LbCas12a system

Expression of LbCas12a protein: We transformed pHMT-LbCas12a into E.coli BL21, and then added 0.2 mM IPTG to induce protein expression (see notebook for details). The result showed that the LbCas12a protein expression in soluble fraction was induced by IPTG, and increased as time goes on (Figure 1). The predicted protein size of LbCas12a with MBP and His-tag is about 180 kDa, which is close to the induced protein indicted by red arrow in figure 1. We also examined the insoluble fraction of IPTG induced BL21 by SDS-PAGE, and confirmed that most LbCas12a protein was soluble (Figure 2).

Fig 1. The Coomassie Blue staining of total protein expression in supernatant of BL21 lysate at different time points.Lane 1: Protein Marker (SMOBIO PM2600) ; lane 2: supernatant of BL21 without IPTG induction; lane 3 ~ 10: supernatant of BL21 induced by 0.2 mM IPTG at 16 °C for 16 hr

Fig 2. The Coomassie blue staining of total protein expression in pellet of BL21 lysate at different time points. Lane 1: Protein Marker (SMOBIO PM2600); lane 2: pellet of cell lysate without IPTG induction; lane 3 ~ 10: pellet of cell lysate induced by 0.2 mM IPTG at 16 °C for 16 hr.

Pre-test of LbCas12a protein purification: After confirming the induction of LbCas12a protein expression, we purify LbCas12a protein by Ni2+-magnetic beads to pull down the His-tag on LbCas12a protein from soluble fraction. The elution of LbCas12a protein from Ni2+-magnetic beads by excess imidazole or TEV enzyme digestion further confirmed that the Ni2+-magnetic beads purification is clear and easy to reverse (Figure 3).

Fig 3. The Coomassie blue staining of protein purification by Ni2+-magnet beads. Lysate: supernatant of cell lysate; flow-through: supernatant before resuspension with Ni2+-magnet beads; TEV treated: treatment of Ni2+-magnet bead-LbCas12a complex by TEV enzyme.

According to the experiments above, we can express and purified LbCas12a proteins for further application. Therefore we scale up the expression and purification of LbCas12a protein.

Large scale protein purification: We use immobilized metal affinity chromatography (IMAC) to purify LbCas12a protein from soluble fraction of BL21 by Ni2+ chelating sepharose column. We then elute LbCas12a protein from Ni2+ column by imidazole and subjected into FPLC separation. The absorption peak at 27-30 fractions was indicated by red arrow in figure 4. To further confirm that the absorption peak, we performed SDS-PAGE and Coomassie blue staining to fractions 27-30, showing that the absorption peak is indeed LbCas12a protein.

Fig 4. Chromatogram of LbCas12a purification by Ni2+ chelating sepharose column. The numbers shown in red indicate the fractions. Each fraction collect 5 ml of wash or elute.

Fig.5. The Coomassie blue staining of elution analyzed by SDS-PAGE. M: marker; L: lysate; FT: flow-through; number: number of fraction tube.