Cloning, expression and purification

1. 1
Under the supervision of :
Dr. Shailja Singh
(Associate Professor)
SCMM, J.N.U, DELHI
Special Centre for Molecular Medicine, Jawaharlal Nehru University,
New Delhi-110067

2. Malaria
 Malaria is a life-threatening disease caused by Plasmodium species.
 There are five parasite species that cause malaria in humans, namely P. falciparum,
P. vivax, P. ovale, P. malariae and P. knowlesi.
 The parasite is transmitted to people through the bite of infected
female Anopheles mosquito
 Malaria is a major health problem in tropical and subtropical region.
 Plasmodium falciparum is the most prevalent malaria parasite in South-East Asia,
accounting for 62.8% of estimated malaria cases in 2017 as reported by WHO.
2
World Malaria Report 2018. Geneva: World Health Organization; 2018

3. 3

4. Life cycle of Plasmodium falciparum
4
David A Fidock et. al DNA Repair Mechanisms and Their Biological Roles in the Malaria
Parasite Plasmodium falciparum

5. Transfer RNA (tRNA)
 tRNA is small well characterized RNA Molecule with a key role in Protein
biosynthesis
 They are directly involved in protein synthesis by carrying amino acids to the
ribosome
 tRNA are key molecules that connect the RNA world and the protein world
 There are specific tRNA for each codon and amino acid
 tRNA are composed of 70-100 nucleotide
 Approximately 20 different types of tRNA are
present
5
Molecular Cell Biology: Seventh Edition, Harvey et al.,
W. H. Freeman & Co., 2013, p135

6. tRNA intron splicing endonuclease
• tRNA Splicing endonuclease is responsible for identification and cleavage of the
Splice site in tRNA
• Splicing reaction is the removal of introns from Pre- tRNA ,This step is catalyzed by
intron Splicing endonuclease
• tRNA molecules may reveal the processes that led to the establishment of the
central dogma: genetic information flows from DNA to RNA to protein.
6Kanai, A .Disrupted tRNA Genes and tRNA Fragments:2015.

7. General Mechanism of tRNA intron Splicing endonuclease
7http://bmb.psu.edu/directory/rch8

8. Objective of the study
 Cloning of tRNA intron splicing endonuclease
 Expression and purification of tRNA intron splicing endonuclease
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9. Materials and Methods
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Chemicals- Acrylamide, Agarose, APS, EDTA, Ampicillin, Etbr, HCL, Sulphuric
Acid, SDS , Nacl, TEMED, Tris , Methanol , Ethanol , Western Blot Marker , anti-
GST IgG-HRP , Glycerol etc.
Plasticware- Microtips , falcon , Petridish, Microcentrifuge tube etc,
Kits- RBC –Real Biotech corporation gel extraction / Plasmid purification kit.
Equipments- Autoclave , B.O.D Incubator , Centrifuge, Deep Freezer , Digital
Balance ,Gel Electrophoresis Apparatus , LAF , Refrigerator , Shaking Incubator ,
Water Bath , Heat Block , PCR , UV- Transilluminator , MQ , Gel Doc , Orbital
Shaker , Oven etc.

10. P.f 3D7_1248000 tRNA-splicing endonuclease, putative
 Gene ID - PF3D7_1248000
 Belong to the Species: Plasmodium falciparum
 Strain: 3D7
 Gene size is 663 bp and protein length is 220 amino acid
 Type: Protein coding
 It is present on chromosome number 12
 Isoelectric point: 4.95
 Number of cysteine present: 5
10

11. Primers used for cloning P.f3D7_1248000 (tRNA intron
splicing endonuclease)
Forward Primer:
ATCggatccATGAAAATTATATATGAATTTAA
Restriction Site - BamH I
Reverse Primer:
GCGgtcgacCTATTTGTATTTATACGATTTGAT
Restriction Site - Sal I
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12. Source: http://ecoliindex.php/pGEX-4T-1
Name : pGEX-4T-1
Description: pGEX- series fusion with Ptac promoter, lac1 repressor, thrombin
Cleavage site ORE; amp resistance enzyme cloning.
Synonyms : pGEX-4T1, pGEX4T-1, pGEX4T1
Type : bacterial plasmid
Form : dsDNA
Size (bp) : 4963
Properties : multiple cloning site, with tag/fusion/marker
Map of pGEX-4T-1 Vector
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13. RESULTS & DISCUSSION
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14. Gene Amplification of Plasmodium falciparum tENDO
Reagent Final Conc. For 20 µl
reaction
10 × Buffer (Taq.) 1 × 2.0 µl
Forward Primer
(100 µm)
10 µM 0.25 µl
Reverse Primer
(100 µm)
10 µM 0.25 µl
dNTPs (100 µm) 20 µM 0.5 µl
Template ( gDNA )
100ng/µl
50 ng 0.5 µl
Taq. DNA
Polymerase
1 Unit 0.25 µl
Mgcl2 (50 mm) 1.5 mM
0.75 µl
NFW - 15.5 µl
141 % agarose Gel
663 bp
Marker PCR Product
700
600

15. Digestion pGEX-4T1 and tRNA intron splicing endo.
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1% agarose gel
4969 bp
663 bp
Reagent Final Conc. For 35 μl
reaction
Insert 50 ng/μl 25 μl
10× Buffer fast
digest
1X 3.5μl
SalI 1 Unit 0.5μl
BamHI 1 Unit 0.5 μl
AMQ 5.5 μl
Reagent Final Conc. For 35 μl
reaction
Pgex4t1 150 ng/μl 10 μl
10× Buffer fast
digest
1X 3.5 μl
SalI 1 Unit 0.5 μl
BamHI 1 Unit 0.5 μl
AMQ 19.99 μl

16. Ligation
• 50 ng of vector was ligated with 1:7 molar ratio
• pGEX4T-1
• Insert tRNA intron Endo
.
• 10× Buffer
• T4 DNA ligase
• Incubate at 16C Over Night
• Transformation into Dh10β cells (E. coli)
16Anderson, S. et al.(1980) A short primer for seq.DNA cloned . Nucleic Acids Res.

17. Colony PCR
Result : Colony-2 is a positive Clone
Ladder C1 C2 C3 Control
663 bp
1% agarose gel
Reagent Final Conc. For 20 µl
reaction
MgCl2 1.5 mM 1.5 mM 0.75 µl
10× Buffer Taq 1x 2.0 µl
Forward Primer 10µm 10µM 0.5µl
Reverse Primer 10µm 10µM 0.5µl
dNTPs 20µm 20µM 0.5µl
Taq DNA Polymerase
(1Unit)
1 Unit 0.25µl
Milli-Q - 15.8 µl
3000
1500
700
100
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18. Restriction digestion
4969 bp
663 bp
1 % agarose Gel
Restriction digestion of
plasmid from positive
clone was done to confirm
the presence of insert in
the plasmid
18

19. 12% SDS Gel
Western Blotting using Anti-GST
HRP antibody
Expression of tRNA intron splicing endonuclease in
E.coli BL21(DE3) cells
19
50 Kda
Coomassie stained SDS gel
250kD
150
50
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20. Protein Extraction and Purification
 Before purifying , the protein needs to be extracted out from the cells.
 The extracted protein needs to be purified in order to separate it from other
types of biomolecules.
 The purified protein is then used for different types of biophysical tests for
different purposes.
Bacterial Culture:
Day 1: Primary culture was inoculated (using E.coli BL21 cells)
Day 2: Secondary culture was inoculated with primary culture and incubated
at 37℃ until the OD reached 0.4-.0.6, then IPTG(1mM) was added to it
for induction and incubated at 37℃ for 12 hours.
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21. Supernatant
Sonication
Centrifugation (protein
present in Supernatant,
pellet)
Supernatant washing
Supernatant Solubilization in
Extraction and Purification
Affinity Chromatography
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22. Purified Protein of tRNA SEN
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23. Conclusion
1. tRNA intron splicing endonuclease (663 bp) was cloned into
the vector (pGEX-4T1) and transformed into the Dh10β cell
(E.coli) and sequencing shows no mutation
2. Protein expression was done in BL21(DE3) cells and
expression was reported in result.
3. tRNA intron splicing endo-nuclease can be used as a probable
drug-target, which will help in developing a different class of
anti-malarials in the near future.
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24. All members of SS Lab J.N.U Delhi
M.Sc Biotech Family 2017-2019
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25. Thank you
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