Difference between revisions of "Part:BBa K3979010"

Latest revision as of 23:44, 20 October 2021

ChiA is from Amycolatopsis orientalis strain B-37

Chitinase A is from Amycolatopsis orientalis strain B-37. The molecular size and weight of the protein are 33.13 kDa and 975 base pairs.

Sequence and Features

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 endochitinase or exochitinase. Endochitinases cleave chitin at internal sites to produce GlcNAc 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[3]. Amycolatopsis orientalis is known to secrete various chitinolytic enzymes such as chitinase β-N-acetylhexosaminidase and endo-β-glucosaminidase. These enzymes have been shown to catalyze efficient transglycosylation activities. It has been reported that the chitinase was able to degrade cell walls of Rhizopus and Mucor which belong to the order of Mucorales more effectively[2].

Experiments

ChiA [BBa_K3979010] is a wild-type chitinase derived from Streptomyces orientalis (also known Amycolatopsis orientalis) strain B-37 . The chitinase was codon optimised for ease of expression in E.coli BL21(DE3) using BOOST. ChiA is an endochitinase capable of hydrolysing and cleaving the (1-4)-beta-linkages in chitin and Chito dextrins. The enzyme has a size of 975 bp and molecular weight of 36.6715 kDa(including 6X His Tag). The approximate PI and extinction coefficient as calculated from ProtParam Tool is 9.39 and 48,150 M^-1 cm^-1 (assuming all cysteine bonds exist).

The wet lab work plan associated with Streptomyces Chitinase (ST) can be broadly divided into three steps:

• Cloning
• Expression and purification

Cloning of Streptomyces Chitinase (ST)

We were able to successfully clone ST into the pET28a vector. The Streptomyces (ST) chitinase 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 uM, respectively.

Fig. 1. ST Forward Primer

Fig. 2. ST Reverse Primer

As the first step, we PCR amplified the ST construct using KOD polymerase. The melting temperature of the construct was 50℃. PCR conditions were standardized with an initial denaturation at 95℃ for 3 minutes, followed by 35 cycles of denaturation (95℃ for 20 seconds), primer annealing (50℃ for 20 seconds), and extension (70℃ for 2 minutes). The final extension was done at 72℃ 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.3).

Fig. 3.Agarose gel run showing successful PCR amplification

Annotations:

1. ST chitinase gene 1(975 bp)
2. ST chitinase gene 1(975bp)

PCR cleanup was then done using the Macherey-Nagel (MN) kit, and the corresponding concentration was 102 ng/μL, measured using nanodrop.

PCR amplified product and pET28a vector were kept for restriction digestion for 6.5 hours. Double digestion was carried out using the enzymes BamHI HF, and HindIII HF received from NEB. A 40μL reaction was set up for a 1μg template in a cutsmart buffer. Double digestion of the vector having BC2 was done using BamHI HF and HindIII HF as the control. Protocols were obtained from NEB double digest calculator. The incubation was done at 37℃ for 6.5 hours. Digested products were then run on agarose gel, and the image of the same is shown below (Figure.4).

Fig.4. Agarose gel run showing successful digestion of ST and cloned BC2 with BC2 and pet28a band

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 17.9 ng/μL and 10.4 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 30mins.

The ligated product was then transformed into competent DH5-alpha cells. We followed a standardized set of protocols for chemical transformation. Along with ligated ST, 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 5)

• 

  Fig5A. DH5alpha E coli. transformed with pet28a containing ST

• 

  Fig5B. DH5alpha E coli. transformed with digested pet28a

• 

  Fig5C. DH5alpha E coli. transformed without any vector

To confirm the transformation, a colony PCR using Taq-polymerase was performed. Six colonies were picked for which, T7 forward primer and T7 terminator reverse primer were used for amplification. PCR conditions were standardized with an initial denaturation at 95℃ for 10 minutes, followed by 20 cycles of denaturation (95℃ for 30 seconds), primer annealing (50℃ for 30 seconds), and extension (72℃ for 3 minutes). The final extension was done at 72℃ for 10 minutes. The expected band size was around 1.3kb, including the 937 kb gene and the T7 promoter and T7 terminator. Cloned BC2 was used as a positive control.(Fig 6)

Fig. 6. Agarose gel run of Colony PCR product of ST

Annotations:

• Colony pcr product of ST cloned in DH5alpha (1.3 kb)
• Positive control - Colony pcr product of BC2 cloned in DH5alpha

Ladder: 1 kb NEB

Four of the 6 selected 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 (Figure 7).

Fig.7. Agarose gel run showing Restriction digestion confirmation of ST cloned in DH5 alpha

Cloned vectors were then transformed into the E.coli BL21(DE3) strain for expression (Figure 8).

• 

  Fig8A. BL21(DE3) E coli. transformed with pet28a containing ST

• 

  Fig8B. BL21(DE3) E coli. transformed with empty pet28a

• 

  Fig8C. BL21(DE3) E coli. transformed without any vector

From the transformed plate, four colonies were picked and restriction digestion was done using BamHI and HindIII to confirm the same. Glycerol stocks were made for the positive colonies.

Fig.9. Agarose gel run showing Restriction digestion confirmation of ST cloned in BL21(DE3)

Fig.10. ST cloned in pET28a

Expression and purification of ST

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 the 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. We conducted a few sets of trial and error experimental conditions which varied from a range of 16℃ to 37℃ and IPTG concentrations of 0.1 mM and 1 mM. From those, we could understand that our protein of interest was getting expressed at 37℃ and 1mM IPTG.

Secondary inoculum (1 litre) induced with 1mM IPTG and incubated for seven hours at 37°C was pelleted down at 11,000 RPM for 20 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 12% SDS-PAGE (Figure 11).

Fig.11. Ni-NTA of Lysate (supernatant) of transformed BL21(DE3) with pet28a cloned with ST

From the gel, it was understood that our protein came only in the pellet obtained after lysis and not in fractions collected after Ni-NTA purification. The lysed pellet had protein with a band size of 36.67 kDa.

Due to lack of time, we couldn’t standardize the purification of native protein via Ni-NTA. We proceeded with urea treatment to expose the His-tag if hidden. For that, 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 followed by a wash with CaCl2 and then pellets were resuspended in an 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 fractions were analysed using SDS-PAGE, strong protein bands were visible in fractions 1 & 3. The gel image is shown below (figure 12).

Fig.12. Ni-NTA of urea treated Lysate (pellet) of transformed BL21(DE3) with pet28a cloned with ST

To the fractions showing protein bands, we added 100% ice-cold ethanol and incubated the solution for 1 hour in -20 degrees 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, 200mM NaCl and 10% glycerol. After purifying the protein, we first measured the concentration of the enzyme using nanodrop. To make the protein concentration accurate, we imputed the protein’s dielectric constant and molecular weight (predicted by ProtParam). Then, we concentrated the protein using an amicon filter to 0.749 mg/ml.

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 threshold binding energy is -6kcal/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.