Part:BBa_K3979003

Bacterial Chitinase Combo 2

Bacterial Chitinase Combo 2 (BC2) (recombinant 2) is a chimeric protein combining domains of ChiA from Amycolatopsis orientalis strain B-37 and ChiB from Serratia marcescens strain QMB1466. The molecular size and weight of the protein are 1161 bp and 39.72 kDa, respectively.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 7
  Illegal XhoI site found at 1156
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 254
  Illegal SapI.rc site found at 15
  

Overview

The chitinase enzyme hydrolyzes insoluble chitin to its oligo and monomeric components. Chitinase proteins are abundant in microorganisms such as bacteria, which use these enzymes to degrade chitin for nutrition. Chitinases are classified as endochitinases or exochitinases. Endochitinases cleave chitin at internal sites to produce GlcNAc (N-acetylglucosamine) multimers. Exochitinases catalyze the progressive hydrolysis of chitin to produce GlcNAc, chitobiose, or chitotriose. Chitinases are classified into different glycoside hydrolase (GH) families based on their amino acid sequences, such as GH18, GH19, and GH20. The GH18 family contains the majority of bacterial chitinases. Based on amino acid sequence homology of the individual catalytic domains, bacterial GH18 chitinases are classified into three major subfamilies: A, B, and C. Chitinolytic bacteria are found in a variety of habitats and decompose chitin in both aerobic and anaerobic conditions. Chitinases from Serratia marcescens are well characterized and found to kill fungi from different genera like Aspergillus, Rhizopus. Tominaga et al. reported that Amycolaptosis orientalis ChiA could lyse the cell wall of fungi like Rhizopus more effectively. Here we are utilizing chitinase domains from both these bacterial species to produce a combinatorial chitinase gene for enhanced activity. The molecular size and weight of the protein are 1161 bp and 39.72 kDa, respectively.

Experiments

Bacterial Chitinase Combo 2 (BC2) [BBa_K3979003] is a hybrid chitinase engineered by combining the catalytic domain of ChiA [BBa_K3979010] from Amycolatopsis orientalis B-37 and the chitin binding domain of ChiB [BBa_K3979009] from Serratia marcescens QMB1466. The chitinase combo has an approximate size of 1.161 kbp and molecular weight of 43.262 kDa(including MW of 6X His Tag). The approximate PI and extinction coefficient as calculated from ProtParam Tool is 9.47 and 73,590 M^-1 cm^-1 (assuming all cysteine bonds exist).

Fig.1. Domain structure of BC2

The wet lab work plan associated with BC2 can be broadly divided into three steps:

• Cloning
• Expression and purification
• Enzyme characterization

Cloning of Bacterial combo 2 (BC2)

We were able to successfully clone BC2 into the pET28a vector. The BC2 construct and corresponding primers were obtained from IDT. Upon receiving the construct and primers, both were dissolved in autoclaved Milli-Q water for a working concentration of 10ng/μL and 100uM respectively, as instructed by the manufacturer. The working stock of the same was 1 ng/μL and 10 μM respectively.

Fig. 2. BC2 Forward Primer

Fig. 3. BC2 Reverse Primer

As the first step, we PCR amplified the BC2 construct using KOD polymerase. The melting temperature of the construct was 50°C;. PCR conditions were standardized with an initial denaturation at 95°C for 3 minutes, followed by 35 cycles of denaturation (95°C; for 20 seconds), primer annealing (50°C for 20 seconds), and extension (70°C for 1 minute). The final extension was done at 72°C for 5 minutes. The reaction volume was set at 50μL. PCR was confirmed by performing a 1% agarose gel electrophoresis. The gel image of the same is shown below:

Fig. 4. Agarose gel run showing successful PCR amplification

PCR cleanup was then done using the Macherey-Nagel (MN) kit and the corresponding concentration was 61.5 ng/μL, measured using nanodrop.

PCR amplified product and pET28a vector were kept for restriction digestion overnight. Double digestion was carried out using the enzymes BamHI HF and HindIII HF received from NEB. A 20μL reaction was set up for a 1μg template in a cutsmart buffer. Single digestion of the vector was done using both BamHI HF and HindIII HF as the control. Protocols were obtained from NEB double digest calculator. The incubation was done at 37°C overnight. Digested products were then run-on agarose gel and the image of the same is shown below (Fig.5).

Fig.5. Agarose gel run showing digested product of BC2 & pet28a

Annotations:

• DD : Double Digestion by BamHI HF and HindIII HF
• SDB : Single Digestion by BamHI HF
• SDH : Single Digestion by HindIII HF

The size of the BC2 band obtained was larger than the expected value. It was observed that the restriction was not successful. Due to the same, experiments were repeated starting from PCR. Added to the reason was the low concentration of purified PCR products.

PCR of BC2 was done again with a slight modification, increasing the extension at 70℃ from 60 seconds to 80 seconds. The rest of the reaction conditions and methods were the same as earlier. A 1% agarose gel electrophoresis was performed. The PCR was successful and the gel image is as shown below (Fig. 6).

Fig. 6. Agarose gel run showing successful PCR amplification

PCR cleanup was done using an MN(Macherey-Nagel) kit. Since earlier the concentration was less, it was decided to elute the PCR cleanup products in a 30 μL elution buffer instead of 60 μL and the eluted concentration was 54.5 ng/μL. Then, we continued to perform restriction digestion. All protocols were kept the same, except the reaction volume and incubation time. The reaction volume was reduced from 50 μL to 40μL and instead of overnight incubation, only 2.5-hour incubation was done. The restriction digestion was successful as shown below (Fig.7).

Fig. 7. Agarose gel run showing digested product of BC2 & pet28a

Gel elution was carried out from the restricted bands using the MN kit using the protocols mentioned in the kit. Concentrations of the vector and insert were 4.9 ng/μL and 65 ng/μL respectively. Using the eluted products, a ligation reaction was carried out. The reaction was set up for 20 μL with T4 DNA ligase. The reaction mixture contained 50 ng of vector and the calculations were made using an in-silico ligation calculator with the molar ratio of vector to insert as 1:3. It was incubated at 23°C for 1 hour.

The ligated product was then transformed into competent DH5α cells. We followed a standardized set of protocols for chemical transformation. Along with ligated BC2, digested vector (negative control1) and competent cells (control2) alone were also kept for transformation and plated. Plates were then incubated at 37°C overnight (Fig 8).

• 

  Fig. 8A. Transformed colonies of cloned BC2 in DH5α competent cells

• 

  Fig. 8B. Transformed colonies of control 2: only competent DH5α cells

• 

  Fig. 8C. Transformed colonies of control 1: digested pet28a in DH5α competent cell

To confirm the transformation, a colony PCR was performed using Taq-polymerase. For the BC2 construct cells and negative controls, T7 forward primer and gene-specific reverse primers were used. The melting temperature of the primers was 50°C and the extension time was set at 2 minutes. Colony PCR couldn't fetch proper results and the gel contained only genomic bands (Figure 9).

Fig. 9. Agarose gel run of Colony PCR product

Next, we did the ligation again and did overnight ligation at 16°C. The ligated vector was then chemically transformed into DH5-alpha cells (Fig. 10)

Fig. 10. Transformed colony of cloned BC2 in DH5α competent cells

The transformed colonies were picked up for digestion confirmation. Plasmid isolation was done using the MN kit, following their protocols. Shown below is the gel image of restriction digestion (Fig. 11)

Fig. 11. Agarose gel run of Digestion confirmation

Annotations:

• Sample 1 : Double digested pet28a using BamH1 HF and HindIII HF
• Sample 2 : Undigested vector
• Sample 3 : Double digested cloned BC2 using BamH1 HF and HindIII HF
• Ladder : NEB 1 Kb Ladder

The plasmids were also verified using Sanger sequencing (Fig. 12)

Fig. 12. Sanger Sequencing result of cloned BC2 (F12_3_06)

Fig. 13. BC2 cloned in pET28a

Cloned vector is then transformed into the E.coli BL21(DE3) strain for expression.

Expression and purification of BC2

Expression

The general procedure followed for expression included a first primary inoculation followed by secondary inoculation, IPTG induction and then SDS. Transformed BL21(DE3) cells were grown in kanamycin-LB-plates and colonies were picked for inoculation. They were inoculated in 10 ml LB media with kanamycin (50 μg /mL) and incubated for 12 hours. It was then added to fresh LB media for secondary inoculation and IPTG was added once the OD value reaches 0.6. After IPTG addition, the culture was incubated and pelleted down. Our first aim was to standardize the amount of IPTG to be added and the incubation time.

For this, we carried out IPTG induction and expression in different sets of IPTG concentrations and temperatures. Our experimental conditions were as follows:

• 35°C 0.1mM IPTG
• 35°C 1mM IPTG
• 37°C 0.1mM IPTG
• 37°C 1mM IPTG
• 30°C 0.1mM IPTG
• 30°C 1mM IPTG
• 26°C 0.1mM IPTG
• 26°C 1mM IPTG
• 16°C 0.1mM IPTG
• 16°C 1mM IPTG

The cells which were pellet down after IPTG induction were lysed and an SDS-PAGE was performed using the lysate (Figure 14). We could understand that our protein was getting expressed in all concentrations of IPTG and temperatures. We could specifically see a broader band in condition number 2, at the required size of 43.262 kDa. Hence, 1mM IPTG and 35°C; were chosen for further experiments downstream.

Fig. 14. SDS PAGE result of IPTG induction and expression at different conditions

Our next aim was to check whether the proteins are expressed in inclusion bodies or not. For this, the cells were induced using 1mM IPTG and kept for incubation at 35°C for 5 hrs. Then the cells were lysed using sonication after adding the lysis buffer (50mM NaPB buffer pH 7, 500mM NaCl, 1mM PMSF, 0.05% beta mercaptoethanol, 5% glycerol, 0.5% triton X-100, 1X protease inhibitor). After centrifugation, both supernatant and pellets were mixed with the loading dye to perform 10% SDS-PAGE along with uninduced control and induced non-sonicated samples (Figure 15).

Fig. 15. SDS PAGE result to check whether proteins are expressed in inclusion bodies or not when induced by 1mM IPTG and incubated at 35°C

Annotations:

1. Ladder- NEB Protein Standard ladder (11-245 kDa)
2. Uninduced sample (Control)
3. Induced sample without lysing the cells
4. Pellet after lysing the induced cells
5. supernatant after lysing the induced cells

We could understand that proteins were forming in the inclusion bodies. So, we decided to induce at a lower temperature of 16°C, since inducing the protein at lower temperature may cause the normal folding of proteins by giving it enough time to fold properly.

Inducing at 16°C followed by lysis using sonication. SDS result shows that protein is forming in cytoplasm as well as inclusion body. (Figure 16)

Fig. 16. SDS PAGE results to check whether proteins are expressed in inclusion bodies or not when induced by 1mM IPTG and incubated at 16°C

Annotations:

1. Sample from Supernatant (After sonication)
2. Sample from Pellet (After Sonication)
3. Sample from Pellet (After Sonication)
4. Sample from Supernatant (After sonication)

The expression of BC2 was thus standardized as mentioned above and our project reached the next goal, purification.

Purification

Secondary inoculum (1 litre) induced with 1mM IPTG and incubated overnight at 16°C was pelleted down at 13,000 RPM for 15 mins. Cells were resuspended in 50 ml lysis buffer (50 mM sodium phosphate buffer (pH 7), 1 mM PMSF, 0.5 M NaCl, 0.05 % BME, 5 % Glycerol, 0.5 % Triton X-100, 1 protease inhibitor tablet) and sonicated in pulse mode (30 seconds ON and 30 seconds OFF for 15 cycles with 55% amplitude). The supernatant was collected and loaded into Ni-NTA column pre-equilibrated with equilibration buffer (50 mM sodium phosphate buffer (pH 7), 1 mM PMSF, 0.5 M NaCl, 0.05 % BME, 5 % Glycerol, 0.5 % Triton X-100, 1 protease inhibitor tablet). It was then washed using 50 mL wash buffer (50 mM sodium phosphate buffer (pH 7), 0.5 mM NaCl, 1 mM EDTA) and eluted in 30 mL elution buffer (50 mM sodium phosphate buffer (pH 7), 0.5 mM NaCl, 1 mM EDTA, 250 mM Imidazole). Collected samples were analyzed using 13% SDS-PAGE (figure 17).

Fig. 17. SDS PAGE result of Ni NTA

Annotations:

• F: Flow Through
• W: Wash Flow through
• E: Elution Flow Through
• 1-20: Elution Fractions

From the gel, it was understood that our protein was coming along with the flowthrough at a band size of 43.262 kDa. We tried elution in NaPB buffer and also tried Ni-NTA from other manufacturers. All these series of experiments suggested that our 6xHis is somehow not able to bind to Ni-NTA, possibly due to its incorporation into protein tertiary structure. After a brief brainstorming session, we decided to perform urea dialysis for purifying BC2.
For performing urea dialysis, the cells were pelleted down after IPTG induction and lysed using lysis buffer (50 mM sodium phosphate buffer (pH 7), 1 mM PMSF, 0.5 M NaCl, 0.05 % BME, 5 % Glycerol, 0.5 % Triton X-100, 1 protease inhibitor tablet). The pellet was collected, washed twice with wash buffer (50mM, NaPB pH7, 0.5mM NaCl, 0.5% Triton X) followed by wash with 10mM CaCl2 and then pellets were resuspended in a extraction buffer containing 8M urea. This was done to denature the protein so as the 6X-His tag will get exposed leading to efficient binding of the protein to the column. After urea treatment, the supernatant was collected and loaded on the Ni NTA resin. When the eluted fraction was analysed using SDS-PAGE, strong bands of the protein were visible. The gel image is shown below: (figure 18).

Fig. 18. SDS PAGE result of Ni NTA

Annotations:

• S: Supernatant
• F: Flow Through
• W: Wash Flow Through
• W: Wash Flow Through
• 1-30: Elution Fractions

We did stepwise dialysis, the denatured protein solution was first brought to equilibrium with a high denaturant concentration (buffer containing 6M Urea), then, the concentration was decreased and brought to equilibrium at a medium concentration (buffer containing 4M Urea) and, then, further decreased and brought to equilibrium at a low concentration (buffer containing 2M Urea). At last, the solution was brought to equilibrium with a buffer containing 0M urea. But the protein was found to be aggregating under prolonged dialysis for removing the urea. Hence, we adopted buffer exchange using protein concentrator columns as a fast method of removing urea. This method was also not successful in providing protein of good concentrations. To overcome this obstacle, we did some literature survey and found that ethanol precipitation method can be used for proteins that are aggregating during urea dialysis.

Following literature, a new batch of protein expression was started. The cells were lysed and the pellet was resuspended in NaPB buffer containing 8M urea. The Ni-NTA column was loaded with this solution and the column was eluted. SDS-PAGE was performed for knowing the fractions containing protein bands. The image of the PAGE gel showing protein bands is shown below: (figure 19).

Fig. 19. SDS PAGE result of Ni NTA

Annotations:

• F: Flow Through
• W: Wash Flow Through
• E: Elution Flow Through
• 1-20: Elution Fractions

To the fractions showing protein bands, we added 100% ice-cold ethanol and incubated the solution for 1 hour in -20 degree celsius. This was followed by centrifugation for bringing down the precipitated protein. The obtained pellet was washed in 90% ethanol and special care was taken to remove the supernatant to the maximum possible extent. The washed pellet was resuspended in 1X PBS containing 0.1% SDS. While concentrating our protein, we did see aggregation when concentrated below 2 mL. Thus, we changed the composition of the buffer by adding stabilizers such as 10% glycerol and increasing the NaCl concentration to 200 mM [Final buffer composition: 1X PBS, 0.1%SDS, 200mM NaCl, and 10% glycerol]. This method was successful in obtaining good concentrations of protein having a tertiary structure, as was confirmed through CD spectroscopy.

Circular Dichroism Spectroscopy

Introduction

Circular Dichroism (CD) is a spectroscopic technique based on the differential absorption of circularly polarised light namely Left-handed Circular (LHC) and Right-handed Circular (RHC). It is exhibited by optically active chiral molecules. CD has various applications such as investigating the charge transfer transitions, geometric and electronic structure etc. ^[1] In the field of Biology, it is primarily used to analyse the different secondary structural types in proteins or polypeptides: alpha helix, parallel and antiparallel beta-sheet, turn etc. ^[2]

Experimental Data

200 µL of 3µM protein solution was used for analyzing the presence of secondary structures such as Alpha Helix and Beta sheets. This was done by measuring the ellipticity at various wavelengths primarily around 222 nm and 208 nm at 20 °C.

Furthermore, we characterized the integrity of the secondary structures with an increase in temperature starting at 20 °C to 100 °C in intervals of 5°C (the enzyme was incubated for 3 mins at each temperature point).

Fig. 20. Ellipticity vs Wavelength

Fig. 21. Ellipticity vs Temperature (208 nm)

Fig. 22. Ellipticity vs Temperature (222 nm)

Results

The graphs were plotted using OriginLab Pro. We observed that the plotted curve for our protein started to significantly deviate from the initial curve at higher temperatures (>90°C). The Ellipticity vs Temperature graph was curve fitted using the Boltzmann Function provided in Origin Lab Pro.

From Fig 21 and 22, it was observed that the beta-sheets were relatively stable at higher temperatures than the Alpha-Helix which had a Tm of about 92.58 °C.

Protein Structure from RaptorX

The annotated sequence was input into the RaptorX server to give us the predicted 3D structure in the form of a PDB file.

Structure

Autodock Results

The receptor, here, is our engineered chimeric chitinase and the ligand is the Chitin octamer(CID 24978517). The threshold binding energy is -6 kcal/mol which is generally accepted as the cut-off in ligand-binding /docking studies, any value more negative than this is considered significant. So, this protein will show binding with the chitin polymer. The protein structures were prepared before docking by removing water molecules, adding polar hydrogen atoms, and adding Kollman charges. A grid box was created so as to eliminate any surface binding and provide us with better and more reliable results. These modifications are necessary for the efficient binding of the ligand to the protein through non-covalent interactions.

Mode	Affinity (kcal/mol)	Dist from RMSD L.B.	Best mode RMSD U.B.
1	-11.8	0	0
2	-11.7	1.287	2.467
3	-10.8	6.245	14.777
4	-10.8	2.900	10.137
5	-10.7	3.885	10.571
6	-10.6	3.525	8.198
7	-10.6	7.961	14.579
8	-10.6	4.779	10.805
9	-10.6	33.198	39.540