Bovine Prolactin Receptor PRLR Gene in expressing Bovine Growth Hormone BGH

1

• Bovine Prolactin Receptor (PRLR) Gene in
  expressing Bovine Growth Hormone (BGH)

2
GROUP E

• Nor Alfarizan Mokhtaruddin 71051
• Najiah Ismail 71050
• Nor Azmi Fahmi 71052
• Jeyanthi a/ p Suppiah 70942
• Liew Lee Kuan 70944
• Asfarina Jamal Mohideen 72595
• Khairul Anuar Mohd Hasan 71048
• Mohamad Fikri Omar 70949

3
OBJECTIVE

• Basically, the objective involve in this cloning
  project includes
• 1) Searching and selecting the gene of
  interest.
• 2) Look for the sequence of the gene.
• 3) Analyze the ORF (open reading frame),
  restriction enzyme site and amino acid
  sequence produced by the gene.
• 4) Compare the sequence of the gene by
  BLAST analysis and multiple DNA sequencing.
• 5) Design the primers for the PCR
  reaction.
• 6) Design cloning strategy which
  involves selecting appropriate vector,
  restriction enzyme, linker, ligase, and
  screening method.
• 7) PCR amplification of the gene.
• 8) Purification of the protein.
• 9) Describing the commercial value of
  the recombinant protein.

4
INTRODUCTION

• The Bovine growth hormone (BGH) also called as
  Bovine Somatotrophin (BST).
• Presents naturally in dairy cows.
• Produced in pituitary glands of cows in small
  amount.
• Increases milk production 10-40.
• Gene involve in production of BGH is Bos taurus
  PRLR gene.

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PROCEDURES IN CLONING STRATEGY

• Step 1 Gene hunting.
• Step 2 Getting the sequence of the gene.
• Step 3 Looking for Open Reading Frame (ORF).
• Step 4Looking for restriction enzyme sites .
• Step 5 BLAST analysis and multiple DNA
  alignment.
• Step 6 designing primer.
• Step 7Selection of vector .
• Step 8 cloning strategy.
• Step 9protein purification.

6
STEP 1 Gene hunting

• Interested to clone the PRLR gene which express
  Bovine Growth Hormone that will enhance milk
  production.
• Accessed the Genbank through http//www.ncbi.nlm.n
  ih.gov to search for it.
• Gene with accession number NM_174155 was
  selected.

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STEP 2 Getting the Sequence of the Gene

• Gene of interest is Bos taurus PRLR.
• Found in NCBI website.
• The gene is linear mRNA.
• Contains 2428 basepair.
• Product of gene recombinant PRLR protein.
• Molecular weight of PRLR
• Since 1 amino acid codes for 1 codon, 1 codon
  contains 3 base pair and 1 amino acids molecular
  weight is 110 Dalton, therefore
• Total amino acid of PRLR gene 2428/3
• Molecular weight PRLR 2428/3 110 D
• 89026.666 D
• 89027 D
• 89 kD

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1 cgggcaaatg ctgaggatac tttccaagtg
aaccctgagt gaacctctaa tatatttatt 61
tcctgtggaa agaggaagga gccaacatga aggaaaatgc
agcatctaga gtggttttca 121 ttttgctact
ttttctcagt gtcagccttc tgaatggaca gtcacctcct
gaaaaaccca 181 agctcgttaa atgtcggtct
cctggaaagg aaacattcac ctgctggtgg gagcctgggg
241 cagatggagg acttcctacc aattacacgc tgacttacca
caaggaagga gaaacactca 301 tccatgaatg
tccagactac aaaaccgggg gccccaactc ctgctacttt
agcaagaagc 361 acacctccat atggaagatg
tacgtcatca cagtaaacgc catcaaccag atgggaatca
421 gttcctcgga tccactttat gtgcacgtga cttacatagt
tgaaccagag cctcctgcaa 481 acctgacttt
ggaattaaaa catccagaag atagaaaacc atatctatgg
ataaaatggt 541 ctccacccac catgactgat
gtaaaatctg gttggttcat tatccagtac gaaattcgat
601 taaaacctga gaaagcaact gattgggaga ctcattttac
tctgaagcaa actcagctta 661 agattttcaa
cttatatcca ggacaaaaat accttgtgca gattcgctgc
aagccagacc 721 atggatactg gagtgagtgg
agcccagaga gctccatcca gatacctaat gacttcccag
781 tgaaggacac aagcatgtgg atctttgtgg ccatcctttc
tgctgtcatc tgtttgatta 841 tggtctgggc
agtggctttg aagggctata gcatggtgac ctgcatcctc
ccaccagttc 901 cagggccaaa aataaaagga
tttgatgttc atctgctgga gatatcacag ccttctcgcc
961 ttgtgtctat gttttaatag gagggcaagt ccgaagaact
tctgcgagct ctggaaagcc 1021 aagacttccc
ccccacttct gactgcgagg acttgctgat ggagttcata
gaggtagatg 1081 actgtgagga ccagcagctg
atgccacgcc cctccaaaga acacacggag caaggcgtga
1141 agcccatgca cctggatctt gacagtgact ctggccgggg
cagctgcgac agcccttcgc 1201 tcttgtctga
aaagtgtgat gaacctcagg cccatccctc caagttccat
actcccgagg 1261 gccctgagaa gctggagaat
ccggaaacaa accttacatg tctccaggcc cctcagagca
1321 caagcgggga aggcaaaatc ccctattttc tggccaatgg
acccaaatct tccacatggc 1381 ctttcccgca
gccccccagc ctatacagcc ccagatattc ttaccacaac
attgctgacg 1441 tgtgtgagct ggccctgggc
atggccggca ccacagccac ttcgctggac caaacagacc
1501 aacatgcttt aaaagcctca aaaaccattg aaactggcag
ggaaggaaag gcaaccaagc 1561 agagggagtc
agaaggctgc agttccaagc ctgaccaaga cacggtgtgg
ccacgacccc 1621 aagacaaaac ccccttgatc
tctgctaaac ccttggaata cgtggagatc cacaaggtca
1681 gccaagatgg agtgctggct ctgttcccaa aacaaaacga
gaagtttggc gcccctgaag 1741 ccagcaagga
gtactcaaag gtgtcccggg tgacagatag caacatcctg
gtattggtgc 1801 cggatccgca agcgcaaaac
ctgactctgt tagaagaacc agccaagaag gccccgccag
1861 ccctgccata gaatccagcc aaggccgacc tggctatctc
ccccacaacc ccaggcaact 1921 gcagactcca
gttgggctgg ggactgggtc ccgcaggttt tatgcactct
tgcagtgaga 1981 gttatggaag gatgggttca
attgtgattt tccttcaggg aacactacag agtacgtgaa
2041 atgcactcta ccagagaggg ctcaagaaca gggttagaat
gacactaccc aactcccagt 2101 tcactcttaa
ttctctattt tcaaccagtt gcctctttgt ccaacagctg
attccagaac 2161 aaatcgttcc atcttgtgtg
atttgtagat ttactttttt gctattagtt gtcagattat
2221 atgttcaaag atataaaagc acattgccta gtattcttaa
gagacagtgc caataggtat 2281 ataatctgga
aaaggccttc atggtttcgt atgtgacaga ggggtataag
tcagtcaaaa 2341 ttgtttacca tgggaagatg
gtagatagga gagaaatgcc atgaaaacca ctttgaagac
2401 cagttgctta acctttgcac tcctcttt
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STEP 3 Looking for ORF

• By surfing http//www.nbci.nlm.nih.gov/gorf/html
• The longest gene segment was to clone.
• Comprises of 891bp start form bases 87 to bases
  977.

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PRLR gene to be cloned

• START CODON
• 87 atgaaggaaaatgcagcatctagagtggttttcattttgctactt
• 132 tttctcagtgtcagccttctgaatggacagtcacctcctgaaaaa
• 177 cccaagctcgttaaatgtcggtctcctggaaaggaaacattcacc
• 222 tgctggtgggagcctggggcagatggaggacttcctaccaattac
• 267 acgctgacttaccacaaggaaggagaaacactcatccatgaatgt
• 312 ccagactacaaaaccgggggccccaactcctgctactttagcaag
• 357 aagcacacctccatatggaagatgtacgtcatcacagtaaacgcc
• 402 atcaaccagatgggaatcagttcctcggatccactttatgtgcac
• 447 gtgacttacatagttgaaccagagcctcctgcaaacctgactttg
• 492 gaattaaaacatccagaagatagaaaaccatatctatggataaaa
• 537 tggtctccacccaccatgactgatgtaaaatctggttggttcatt
• 582 atccagtacgaaattcgattaaaacctgagaaagcaactgattgg
• 627 gagactcattttactctgaagcaaactcagcttaagattttcaac
• 672 ttatatccaggacaaaaataccttgtgcagattcgctgcaagcca
• 717 gaccatggatactggagtgagtggagcccagagagctccatccag
• 762 atacctaatgacttcccagtgaaggacacaagcatgtggatcttt
• 807 gtggccatcctttctgctgtcatctgtttgattatggtctgggca
• 852 gtggctttgaagggctatagcatggtgacctgcatcctcccacca

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STEP 4 Looking for restriction enzyme site

• Analyze the sequence for common restriction
  enzyme site at Webcutter through
  http//www.firstmarket.com/cutter/cut2.html .
• Search for endonucleases that do not cut the gene
  of interest.
• So that, an endonuclease can be selected to cut
  the DNA sequence and the vector as well at same
  restriction site.

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• The following endonucleases were selected but
  don't cut this sequence
• AatII, Acc16I, Acc65I, AccBSI, AccI, AclNI,
  AfeI, AgeI, AhdI, Aor51HI, AscI, AseI, AsnI,
  Asp718I, AspEI, AviII, AvrII, BanIII, BbuI, BcgI,
  BclI, BglII, BlnI, BlpI, Bpu1102I, Bpu14I,
  Bsa29I, BsaMI, BsaOI, BscI, BseCI, BsePI,
  Bsh1285I, BsiEI, BsiI, BsiWI, BsmBI, BsmI,
  Bsp106I, Bsp119I, Bsp1407I, Bsp1720I, Bsp68I,
  BspCI, BspDI, BspHI, BspXI, BsrBI, BsrGI, BssHII,
  BssSI, Bst1107I, BstBI, BstD102I, BstMCI, BstSNI,
  BstZI, Bsu15I, CciNI, CelII, Cfr42I, ClaI, CpoI,
  Csp45I, CspI, DrdI, EagI, Eam1104I, Eam1105I,
  EarI, EclHKI, EclXI, Eco105I, Eco47III, Eco52I,
  EcoNI, EcoRI, EcoT22I, Esp3I, FbaI, FseI, FspI,
  HincII, HindII, HindIII, HpaI, KpnI, Ksp22I,
  Ksp632I, KspI, LspI, MluI, Mph1103I, Mva1269I,
  NheI, NotI, NruI, NsiI, NspV, PacI, PaeI, PaeR7I,
  Pfl23II, PinAI, Ple19I, PmeI, Ppu10I, PshAI,
  PshBI, Psp1406I, PspLI, PstNHI, PvuI, RcaI,
  RsrII, SacII, SalI, SapI, SbfI, SexAI, SfiI,
  Sfr274I, Sfr303I, SfuI, SgfI, SgrAI, SmiI, SnaBI,
  SpeI, SphI, SplI, SrfI, Sse8387I, SspBI, SspI,
  SstII, SunI, SwaI, VspI, XhoI, XmaIII, Zsp2I
• Out of all the endonucleases that had been
  suggested, we used EcoR1 as our restriction
  enzyme for the cloning purpose

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STEP 5 (a) Compare the sequence with BLAST
analysis

• Compare the PRLR gene with other gene sequences
  in single form by showing the degree of
  similarities.
• Detect relationship among sequences.
• Result

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• The score of each alignment indicated by one of
  five different colours.
• Result of comparing PRLR with gi31342717refNM_1
  74155.2 Bos taurus prolactin receptor (PRLR),
  mRNA

Query 1 cgggcaaatgctgaggatactttccaagtgaaccctga
gtgaacctctaatatatttatt 60

Sbjct 1 cgggcaaatgctgaggatactttccaagt
gaaccctgagtgaacctctaatatatttatt 60
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(b) Multiple DNA sequence alignment

• Done by using CLUSTALW program.
• Produces multiple sequence alignment of divergent
  sequences within different organisms.
• Result
• NM_174155.2 CGGGCAAATGCTGAGGATACTTTCCAAGTGAACCCT
  GAGTGAACCTCTAATATATTTATT 60
• AF027403.1 CGGGCAAATGCTGAGGATACTTTCCAAGTGAACCCT
  GAGTGAACCTCTAATATATTTATT 60
• PRLR gene CGGGCAAATGCTGAGGATACTTTCCAAGTGAACCCT
  GAGTGAACCTCTAATATATTTATT 60
• AF416619.1 ------------------------------------
  ------------------------
• NM_174155.2 TCCTGTGGAAAGAGGAAGGAGCCAACATGAAGGAA
  AATGCAGCATCTAGAGTGGTTTTCA 120
• AF027403.1 TCCTGTGGAAAGAGGAAGGAGCCAACATGAAGGAA
  AATGCAGCATCTAGAGTGGTTTTCA 120
• PRLR gene TCCTGTGGAAAGAGGAAGGAGCCAACATGAAGGAA
  AATGCAGCATCTAGAGTGGTTTTCA 120
• AF416619.1 -------------------AGCCAACATGAAGGAA
  AATGTGGCATCTGCAACCGTTTTCA 41
• 
  

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• Also indicates evolutionary relationship through
  filogram/ cladogram.
• Results of comparing PRLR with AF027403.1,
  NM_174155.2, AF416619.1

PRLR gene
AF416619.1
NM_174155.2
AF027403.1
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STEP 6 Design primer for PCR amplification

• Two primers involve (a) forward primer.
• (b)
  reverse primer.
• Criteria of primer
• Usually primers used are 18 to 30bp in length.
• Forward primer should not be complementary with
  reverse primer.
• The primers should not repeated sequences to
  prevent self complementary of the primer which
  will form a hairpin loop structure.
• The 3end of the primer should be complementary
  to the target DNA sequence, 5end can have any
  other sequences.
• The distance between primer should be less than
  10kb in length.

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• From http//www.invitrogen.com/content.cfm?pageid
  9716
• The designed primers are
• forward primer 5 atgaaggaaaatgcagcatctag 3
• reverse primer 5 ttaaaacatagacacaaggcgag 3
• Both forward and reverse strands consist of
  39.13 of GC in their sequence.
• Melting temperature
• forward primer, Tm 59.77
• reverse primer, Tm 58.51
• Lower temperature was chosen as the annealing
  temperature for PCR 58.51C

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STEP 7 Selection of vector

• Selected vector pRSET B
• Criteria of pRSET
• High-level expression from the bacteriophage T7
  promoter.
• T7 gene 10 sequence to provide protein stability.
• N-terminal polyhistidine (6xHis) Tag for rapid
  purification with ProBond resin.
• N-terminal Xpress epitope for protein detection
  with the Anti-Xpress Antibody.
• Enterokinase cleavage site for removal of fusion
  Tag.
• f1 origin for ssDNA rescue to allow easy
  sequencing and mutagenesis.
• Selected host BL21 (DE3) E.coli contains a
  chromosomal copy of the T7 DNA polymerase gene

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Vector
21
Genetics Markers for pRSET B vector
2887 nucleotides T7 promoter bases 20
39 6xHis Tag bases 112 129 Anti-XpressTM
epitope bases 169 192 Multiple
cloning site bases 202 248 f1 origin
bases 456 911 Ampicilin resistant gene bases
1042 - 1902 ColE1 bases 2196 - 2552
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STEP 8 CLONING STRATEGY
Source Bos Taurus (cow
5
3
pRSET B 2.9kb
pRSET B 2.9kb
Bovine prolactin receptor Gene ( mRNA )
vector
reverse transcriptase
cut
5
3
cDNA
with
3OH
EcoR1
T4 DNA Polymerase
5P
5
3
dsDNA
5P
5
3
3OH
adapter/linker and cut with EcoR1
3OH
Calf Intestine
3OH
5P
5OH
Alkaline
3OH
5P
Phosphatase
3OH
5OH
T4 DNA ligase

Recombinant protein
Transform into E.coli host
Grow on agar plate (AMPX-GAL)
Screening through PCR
Purified recombinant protein
Protein purification
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(8.1) PCR AMPLIFICATION OF PRLR GENE

• Purpose is to produce more DNA for further study.
• Using a heat-stable DNA polymerase (Taq DNA
  polymerase) and an automated thermal cycler (PCR
  machine).
• Components for PCR amplification
• forward primer 5atgaaggaaaatgcagcatctag3
• reverse primer 5ttaaaacatagacacaaggcgag3
• Taq polymerase
• Template
• MgCl2
• PCR buffer
• dNTPs
• distilled water
• Annealing temperature 58.51C

24
(No Transcript)
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(8.2) Adding EcoR1 linker to PRLR

• To ligate with Taq polymerase PCR product which
  have an adenine residue (A) at the end.
• EcoR1 linker designed with a thymine (T) at the
  end
• so that later it can anneal and ligate to our DNA
  of interested by using ligase
• cut by the EcoR1 enzyme to produce sticky ends
  which complementary with the vector cut with same
  enzyme.

EcoR1 linker
5
3
5
3
A
A T
T A
A
PRLR with EcoR1 linker
PRLR
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(8.3) Ligation

• Process of joining linear DNA fragment with
  covalent bonds.
• A phosphodiester bond created.
• Selected ligase T4 DNA ligase.
• EcoR1 enzyme used to cut the PRLR gene with EcoR1
  linkers and pRSET B vector.

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Ligation

• Before ligation, vector treated with CIAP (calf
  intestinal Alkaline Phosphatase) to prevent
  self-ligation and recircularization of vector.
• Ligation of PRLR gene and pRSET B produced a
  recombinant gene with 3778bp.

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• Genetic Markers for the recombinant gene

PRLR gene bases 227 1117 T7 promoter bases
20 39 6xHis Tag bases 112 129 Anti-XpressTM
epitope bases 169 192 f1 origin bases
1346 1801 Ampicilin resistant gene bases
1932 - 2792 ColE1 bases 3086 3442
pRSET B-PRLR
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(8.4) Transformation

• Transformation transfer of a recombinant plasmid
  DNA into a host cell.
• Using calcium chloride method.
• To determine the success of transformation
• culture E.coli cell on the nutrients agar
  plate with ampicillin.
• The ampicillin resistance gene in pRSET, protects
  the E.coli from the ampicillin.
• Colony appear on the plate selected for screening
  method.

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(8.5) Screening of recombinant clone by PCR

• The cloning method applied is non directional
  cloning method.
• To confirm the presence of PRLR gene in the pRSET
  B vector
• Components for PCR screening
• dNTPs
• PCR buffer.
• MgCl2
• Taq DNA polymerase.
• T7 primer 5GGAGATATACATATGCGGGGTTCT3
• Reverse primer 5TTAAAACATAGACACAAGGCGAG3
• Sterile deionised distilled water.
• There are possibilities of getting PRLR inserted
  in 2 orientation.

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Orientations
PRLR gene in forward orientation
32
PRLR gene in reverse orientation
33
(No Transcript)
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Protein purification

• based on the remarkable affinity between the
  6xHis Tag and divalent nickel cations.
• PRLR gene protein with His Tag bound to nickel on
  the surface of resin.
• other protein and substances of E.coli were
  washed.
• released the PRLR gene protein from His Tag by
  cleaved it with enterokinase at its enterokinase
  cleavage site.
• Purified protein was produced

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(9.1) Protein screening by using SDS-PAGE

• Study of the movement of charge molecule in an
  electric field.
• Medium Polyacrylamide.
• Buffer Laemli buffer.
• Sample buffer
• Sodium didocyl sulfate (SDS).
• Beta mercapethanol.
• Glycerol.
• Bromo phenol blue dye.
• Visualizer Coomassie brilliant blue.
• Band showed 89 kD indicate the protein coded by
  PRLR gene was seen.

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Commercial values

• Dairy products.
• Cosmetic products.
• Pharmaceutical products.

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References

• http//mend.endojournals.org/cgi/content/abstract/
  11/10/1449
• http//endo.endojournals.org/cgi/full/138/8/187
• http//www.nelh.hns.uk/hth/milk_stomach.asp
• http//physrev.physiology.org/cgi/content/full/80/
  4/1523SEC2.1
• http//helies.bto.ed.ac.uk/bto/glossary/no.htm
• www.biowed.uwlax.edu/Genweb/Molecular/Seq_Anal/Tra
  nslation/translation
• http//www.ncbi.nlm.nih.gov
• http//www.firstmarket.com/cutter/cut2.html
• http//www2.ebi.ac.uk/clustalw/
• http//www.genome.wi.mit.edu/cgi-bin/primer/primer
  3_www.cgi
• http//www.nbci.nml.nih.gov/gorf/gorf.html
• Invitrogen 1997 product catalog, 112, 237.
• STRATAGENE (Tools And Technology For Life
  Sciences. 2001/2002 CATALOG.