1. Jayanti Tokas1, Rubina Begum1, Shalini Jain2 and Hariom Yadav2
INTEIN MEDIATED
PROTEIN SPLICING
Jayanti Tokas1, Rubina Begum1, Shalini Jain2 and Hariom Yadav2
1Department of Biotechnology, JMIT, Radaur
2 NIDDK, National Institute of Health, Bethesda,MD20892, USA

2. PROTEIN SPLICING Self catalyzed protein rearrangement
Inactive protein precursor to biologically active protein
Post translational cellular process
Intervening sequences - INTEIN is excised out
Flanking sequences - EXTEINS are ligated
Altered the “central dogma” of gene expression

3. Intein Mediated Protein Splicing Occurs Post-translationally

4. Protein Splicing
RNA Splicing

5. Discovery 73% 77% N domain Spacer domain C domain
In 1990,in Saccharomyces cerevisiae vacuolar ATPase
The TFP1 gene of S. cerevisiae encodes two proteins.
69kd catalytic subunit of V type ATPase
50kD protein.
73% %
N domain Spacer domain C domain
Homology maps to N and C terminal regions
TFP1 gene encodes 1071 a.a,119 kD protein which show
homology to catalytic subunit of v type ATPase of neurospora. 1
273
608
Neurospora vma-1
Yeast TFP1 1
738
284
1071

6. X-XbaI,B-BamHI,E-EcoRV,H-HindIII,K-KpnI,N-NacI,P-PstI,S-SacII,Sp-SphI
Restriction fragments of TFP1 were placed under an inducible promoter,expressed in E.coli and immunoblotted using mAbs to 69kD subunit
None of the Abs recognized the spacer domain
N and C domains are joined to form 69kD subunit N S X B E P K H Sp X
ATG
stop
X-XbaI,B-BamHI,E-EcoRV,H-HindIII,K-KpnI,N-NacI,P-PstI,S-SacII,Sp-SphI

7. What are Inteins ???
Genetic elements that disrupt the coding sequence of genes.
Transcribed & translated together with their host protein.
Found in organisms of all 3 domains of life:
Eucaryotes(70 inteins)
Eubacteria (150 inteins)
Archaea(110 inteins)
and in viral and phage proteins.
Uptil now over 350 inteins have been described.
Found in metabolic enzymes, DNA and RNA polymerases,
proteases, ribonucleotide reductases and V-type ATPase.

8. Inteins consisting of only the self splicing domain are
Inteins consists of two domains.
* Splicing domain which comprises the N and C terminals.
* An endonuclease domain.
Conserved Motifs
Inteins consisting of only the self splicing domain are
called mini-inteins.

9. A pair of split DnaE genes produces two precursor polypeptides.
Split inteins: eg. In DnaE, the catalytic subunit of DNA polIII in Nostoc Punctiforme
A pair of split DnaE genes produces two precursor polypeptides.
dnaE-n
dnaE-c
Transcription and Translation
N-intein
C-intein N C
Inactive Precursors
Trans Protein Splicing
Active DNA Pol cat. subunit N C

10. Intein protein splicing domain
Endonuclease N C N C C N
Intein protein splicing domain
(embedded in Myc. leprae gyrase gene)
Intein protein splicing domain with homing endonuclease
(embedded in S. cerevisiae proton ATPase)
Intein protein splicing domain with homing endonuclease--DNA bound

11. VMA intein-Vacuolar Membrane, ATPase intein
Structure of PI-SceI
454 amino acid(~50kD )protein encoded by the intervening sequence of TFP1.
Composed of 2 domains:
~a splicing domain &
~an endonuclease domain.
Domain I comprises the first 182 & last 44 residues.
Compact & globular domain II.
comprises the residues.
VMA intein-Vacuolar Membrane, ATPase intein

12. An extreme example from a marine cyanobacterium…
RIR is RNR…ribonucleotide reductase

13. Homing Endonuclease Activity
Homing is the lateral transfer of an intervening sequence to a homologous allele that lacks the sequence.
Catalysed by an endonuclease that recognizes and cleaves the target allele.
Most endonucleases have a conserved DOD sequence(LAGLIDADG)

14. Intein mobility (homing)
Inteins as parasitic genes
Gene conversion process via double strand break repair I+ I+ I-
= extein
= intein
= homing endonuclease
Homing endonucleases give intervening sequences the ability to be invasive and persistant

16. The Mechanism of Protein Splicing

17. The Mechanism of Protein Splicing
Step 1: Formation of an ester intermediate by an N-O acyl
rearrangement of the conserved Ser /Cys residue at the
upstream splice junction. Step 2: Formation of a branched intermediate by transesterification
Step 3: Intein excision by peptide bond cleavage coupled to
succinimide formation involving the conserved Asn residue
at the downstream splice junction. Step 4: Spontaneous  O-N acyl rearrangement in formation of peptide
bond between 2 exteins.

18. The Mechanism of Protein Splicing
Step 1 Acyl N/O shift (or N/S)

19. The Mechanism of Protein Splicing
Step 2 Transesterification

20. The Mechanism of Protein Splicing
Step 3 Asn cyclization/succinamide formation

21. The Mechanism of Protein Splicing
Step 4 Acyl O/N shift (or S/N) & Succinimide hydrolysis

22. The Mechanism of Protein Splicing

23. Variations in Splicing Pathway
The ribonucleotide reductase large subunit of the Chilo iridescent virus (CIV) contains an intein having a C terminal Glutamine instead of Asn.

24. An unique RNR intein from Carboxydothermus hydrogenoformans(Chy) has a C terminal Aspartate.

25. K1bA inteins, Mycobacterial DnaB inteins and Deinococcus
radiodurans Snf2 inteins begin with Alanine (N-terminal) instead of Ser1 or Cys1.

26. Applications of protein splicing
Rapid purification of target proteins
Temperature sensitive control of protein activity by conditionally splicing inteins.

27. Rapid purification of target proteins
Affinity purification using a tag
Affinity purification using a modified intein tag
affinity tag
target protein
affinity purification
affinity tag
target protein
elution
affinity tag
target protein
Protease
treatment
protease
affinity tag
target protein
further purification
target protein

28. Temperature sensitive control of protein activity by conditionally splicing inteins.
Temperature sensitive splicing variants of VMA intein can be inserted within transcription factors that can be active only at temperatures at which splicing occurs. I
Gal4 AD
29ºC: No splicing
18ºC: Splicing
Gal4 AD
Gal4 BD I
Gal4 BD
Target
Target
UAS
UAS
Gal80
29ºC: No Splicing I I
Gal80
Gal4 AD
Gal4 AD
Gal4 BD
Gal4 BD
Target
Target
UAS
UAS
18ºC: Splicing
(Nature BT, 2004)

30. Thank you