P1251918558Zhusq

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CLONING SIMULATION PROJECT 2003 Cloning of Human
Aldolase B gene Towards the Application in
Hereditary Fructose Intolerance(HFI) By Group A
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• HO YI CHIEW
• ABD.RASHID B.JUSOH
• SITI FATIHAH ARIFFIN
• MUHAMMAD ZAKI
• B.ABDUL AZIZ

• EDINUR HISHAM
• B.ATAN
• SITI SALWA
• BT.KASSIM
• ABDUL HAIL AHMAD
• TARMIZZI

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OBJECTIVES

• To understand the concepts of molecular biology
  technique.
• To understand the application of recombinant DNA
  technology.
• To understand the functions of the recombinant
  protein.
• To learn how to design a relevant cloning
  strategy.

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INTRODUCTION ALDOLASE B GENE
STRUCTURE OF ALDOLASE B GENE
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WHAT IS ALDOLASE B GENE?

• -encodes for aldolase B enzyme
• ALDOLASE B ENZYME
• -major aldolase isozymes in the liver
• -specialized functions in
• fructose metabolism using F-1-P (as
  substrate)
• gluconeogenesis produce F-1,6-BP from
• G3P and DHAP

Mutations in the human Aldolase B gene that
result in diminished aldolase B activity cause
the autosomal recessive disease, Hereditary
Fructose Intolerance (HFI)
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Normal Fructose Metabolism
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Aldolase B enzyme

• - class I aldolases
• - tetrameric
• - glycolytic enzyme
• - catalyzes reversible cleavage of F-1,6-BP to
  G3P and DHAP.
• - catalyzes cleavage of structurally related
  sugar phosphates including F1P, an intermediate
  of fructose metabolism.

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Fructose Metabolism in HFI
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Pathophysiology of HFI

• - inability of aldolase B to cleave F1P
  effectively
• creates a build up of F1P.
• - inorganic phosphate(Pi) which is used to
• regenerate ATP is sequestered in the
• accumulating F1P.
• 1) Hypophasphatemia
• 2) Increase in serum magnesium (Mg)
• 3) Hyperuricemia
• 4) Secondary hypoglycemia

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Hereditary Fructose Intolerance (HFI)

• ? A genetic disorder since 1956.
• Results from a deficiency of aldolase B
• activity in the
• liver, kidney and intestine.
• most dire for the newborn infant
• period of greatest risk - weaning during infancy
  
• ? Symptoms of HFI
• -abdominal pain
• -vomiting
• -hyperglycemia
• ? incidence rate - 1 in 22,000 (most recent
  study)

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How is HFI diagnosed?

• 1) An enzymatic assay to determine aldolase
  activity.
• 2) A fructose tolerance test.
• Is there a Cure?
• HFI is a heritable genetic disease.
• -a cure would involve replacement of the gene
  or of the
• lost aldolase B enzyme activity directly to
  the liver,
• kidney, and intestine.
• -Currently, there are no procedures that can
  do this.

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STEPS IN CLONING SIMULATION PROJECT
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Step 1 Hunt for gene and get the
gene sequence

• ? We visited Genbank website
• http//www.ncbi.nlm.nih.gov to get the gene
• sequence.
• ? Accession number NM_000035.
• ? Definition Homo sapiens aldolase B,
• fructose-biphosphate (ALDOB).
• 1654 base pair in length
• version NM_000035.1 GI 4557306.
• ? Coding sequence (CDS) region of base pairs
• between 126-1220 bp.
• ? The sequence is

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Homo sapiens aldolase B, fructose-bisphosphate
(ALDOB), mRNA. BASE COUNT 470 a 420 c
393 g 371 t 1 aaaaacatga tgagaagtct
ataaaaattg tgtgctacca aagatctgtc ttatttggca 61
gctgctgcct cacccacagc ttttgatatc taggaggact
cttctctccc aaactacctg 121 tcaccatggc ccaccgattt
ccagccctca cccaggagca gaagaaggag ctctcagaaa 181
ttgcccagag cattgttgcc aatggaaagg ggatcctggc
tgcagatgaa tctgtaggta 241 ccatggggaa ccgcctgcag
aggatcaagg tggaaaacac tgaagagaac cgccggcagt 301
tccgagaaat cctcttctct gtggacagtt ccatcaacca
gagcatcggg ggtgtgatcc 361 ttttccacga gaccctctac
cagaaggaca gccagggaaa gctgttcaga aacatcctca 421
aggaaaaggg gatcgtggtg ggaatcaagt tagaccaagg
aggtgctcct cttgcaggaa 481 caaacaaaga aaccaccatt
caagggcttg atggcctctc agagcgctgt gctcagtaca 541
agaaagatgg tgttgacttt gggaagtggc gtgctgtgct
gaggattgcc gaccagtgtc 601 catccagcct cgctatccag
gaaaacgcca acgccctggc tcgctacgcc agcatctgtc 661
agcagaatgg actggtacct attgttgaac cagaggtaat
tcctgatgga gaccatgacc 721 tggaacactg ccagtatgtt
actgagaagg tcctggctgc tgtctacaag gccctgaatg 781
accatcatgt ttacctggag ggcaccctgc taaagcccaa
catggtgact gctggacatg 841 cctgcaccaa gaagtatact
ccagaacaag tagctatggc caccgtaaca gctctccacc 901
gtactgttcc tgcagctgtt cctggcatct gctttttgtc
tggtggcatg agtgaagagg 961 atgccactct caacctcaat
gctatcaacc tttgccctct accaaagccc tggaaactaa 1021
gtttctctta tggacgggcc ctgcaggcag tagcactggc
tgcctggggt ggcaaggctg 1081 caaacaagga ggcaacccag
gaggctttta tgaagcgggc catggctaac tgccaggcgg 1141
ccaaaggaca gtatgttcac acgggttctt ctggggctgc
ttccacccag tcgctcttca 1201 cagcctgcta tacctactag
ggtccaatgc ccgccagcct agctccagtg cttctagtag 1261
gagggctgaa agggagcaac ttttcctcca atcctggaaa
ttcgacacaa ttagatttga 1321 actcgctgga aatacaacac
atgttaaatc ttaagtacaa gggggaaaaa ataaatcagt 1381
tatttgaaac ataaaaatga ataccaagga cctgatcaaa
tttcacacag cagtttcctt 1441 gcaacacttt cagctcccca
tgctccagaa tacccaccca agaaaataat aggctttaaa 1501
acaatatcgg ctcctcatcc aaagaacaac tgctgattga
aacacctcat tagctgagtg 1561 tagagaagtg catcttatga
aacagtctta gcagtggtag gttgggaagg agatagctgc 1621
aaccaaaaaa gaaataaata ttctataaac cttc
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Step 2 Open Reading Frame (ORF) Analysis

• ? ORF is a DNA sequence that contains series of
  codons,
• which can be translated into protein.
• ? Must have start codon (ATG) and ends with a
  stop codon
• (TGA,TAG or TAA).
• ? make sure that the interested gene region can
  be expressed.
• ? To analyze the open reading frame, we used the
  website
• http//www.ncbi.nlm.nih.gov/gorf/gorf.htm
• From the ORF results, we choose 3 frame(126bp
  1220bp,
• 1095 bp in length)
• 3 frame has start codon and stop codon.

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ORF RESULT

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Frame From to Length
3 126 1220 1095
-1 35 345 312
-2 319 788 270
3 1227 1394 168
-1 791 949 159
-2 445 603 159
-1 1217 1342 126
-1 377 499 123
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Step 3 Restriction Enzyme Analysis
Restriction enzyme is a protein which recognized
specific short nucleotide sequences and will cut
at those sequences to give fragments of DNA. In
order to analyze the sequence for common
restriction enzyme site for Aldolase B
gene, accesed website http//www.firstmarket.com/
cutter/cut2.html No restriction endonuclease
enzyme is chosen to cut our gene of interest
Two restriction enzymes have been chosen to cut
the vector EcoRI and HindIII Both enzymes do
not cut the desired gene sequence.
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The following endonucleases were selected but
don't cut this sequenceAatI, AatII, Acc113I,
Acc16I, AccBSI, AccIII, AclNI, AcyI, AgeI,
AhdI,Alw44I, Ama87I, AocI, ApaLI, AscI, AseI,
AsnI, Asp700I, AspEI, AspI, AtsI,AvaI, AviII,
AvrII, BanIII, BbeI, BbiII, BbrPI, BbsI, BbuI,
Bbv16II, BcgI,BcoI, BlnI, BlpI, BpiI, Bpu1102I,
Bpu14I, BpuAI, Bsa29I, BsaAI, BsaBI,BsaHI,
BsaMI, BsaOI, BsaWI, BscI, Bse21I, Bse8I, BseAI,
BseCI, BsePI, Bsh1285I,Bsh1365I, BsiEI, BsiMI,
BsiWI, BsmBI, BsmI, BsoBI, Bsp106I, Bsp119I,
Bsp13I,Bsp1407I, Bsp1720I, Bsp68I, BspCI, BspDI,
BspEI, BspHI, BspMI, BspXI, BsrBI,BsrBRI, BsrDI,
BsrGI, BssHII, BstBI, BstD102I, BstEII, BstMCI,
BstPI, BstSNI,BstZI, Bsu15I, Bsu36I, CciNI,
CelII, Cfr42I, Cfr9I, ClaI, CpoI, Csp45I,CspI,
CvnI, DraIII, DrdI, EagI, Eam1105I, EclHKI,
EclXI, Eco105I, Eco147I,Eco255I, Eco52I, Eco72I,
Eco81I, Eco88I, Eco91I, EcoO65I, EcoRI,
EcoT22I,EheI, Esp3I, FauNDI, FseI, FspI, Hin1I,
HindIII, HpaI, Hsp92I, KasI, Kpn2I,KspI, LspI,
MamI, MfeI, MluI, Mph1103I, MroI, Msp17I, MunI,
Mva1269I, NarI,NdeI, NheI, NotI, NruI, NsiI,
NspV, PacI, PaeI, PaeR7I, Pfl23II, PinAI,Ple19I,
PmaCI, Pme55I, PmeI, PmlI, Ppu10I, PshBI,
Psp1406I, PspAI, PspALI,PspEI, PspLI, PstNHI,
PvuI, RcaI, RsrII, SacII, SalI, ScaI, SexAI,
SfiI,Sfr274I, Sfr303I, SfuI, SgfI, SgrAI, SmaI,
SmiI, SnaBI, SpeI, SphI, SplI,SrfI, SseBI,
SspBI, SstII, StuI, SunI, SwaI, Tth111I, VneI,
VspI, XbaI,XcmI, XhoI, XmaI, XmaIII, XmnI, Zsp2I
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Step 4 Determining of amino acid sequence and
molecular weight
Determination of amino acid sequence of aldolase
B gene - by using Bioedit software Length
1654 base pairs Molecular Weight 504410
Daltons, single stranded Molecular Weight
1005265 Daltons, double stranded GC content
49,15 AT content 50,85 Nucleotide Number
Mol A 470 28,42 C
420 25,39 G
393 23,76 T 371
22,43
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Molecular weight of 3 frame ORF of aldose B gene
1095 x 110 Dalton 3 40150
Dalton 40.15 kDa approximately 40 kDa
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STEP 5 Compare the Gene Sequence With Other
Similar Gene Sequencesa) BLAST
Analysis (Basic Local Alignment Search Tool)

• BLAST
• - program designed for comparison of the
  protein or gene of interest
• with all the available sequence databases
  that share the similarity.
• - uses a heuristic algorithm which seeks local
  as opposed to global
• alignments.
• - able to detect relationships among sequences
  which share the
• isolated regions of similarity.
• NCBI website http//www.ncbi.nlm.nih.gov
• Score of each alignment - indicated by one of
  five different colors
• -
  divides the range of scores into five groups
• Each alignment represents an organism.
• Expected value(E) the degree of similarity.
• E 0, means the compared gene sequences is 100
  identical to
• each other.

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• These are some of the definitions and scores with
• associated alignments
• gi4557306refNM_000035.1 Homo sapiens
  aldolase B, fructose-bisphosphate
• (ALDOB), mRNA.
• Length 1654
• Score 3279 bits (1654), Expect 0.0
• Identities 1654/1654 (100)
• Strand Plus / Plus
• 2) gi443800embZ29372.1OAMRALDB O.aries mRNA
  for aldolase B
• Length 1649
• Score 1443 bits (728), Expect 0.0
• Identities 1057/1163 (90), Gaps 4/1163
  (0)
• Strand Plus / Plus

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3) gi23271468gbBC024056.1 Mus musculus
aldolase 2, B isoform, mRNA(cDNA clone
MGC36391 IMAGE5100067), complete cds
Length 1971 Score 1354 bits (683), Expect
0.0 Identities 1000/1103 (90), Gaps
2/1103 (0) Strand Plus / Plus 4)
gi27476058refNM_012496.1 Rattus norvegicus
aldolase B (Aldob), mRNA Length 1547
Score 1211 bits (611), Expect 0.0
Identities 985/1107 (88), Gaps 2/1107 (0)
Strand Plus / Plus 5) gi202839gbM10149.1
RATALDBC Rat liver aldolase B mRNA, complete
cds Length 1547 Score 1211 bits
(611), Expect 0.0 Identities 985/1107
(88), Gaps 2/1107 (0) Strand Plus /
Plus
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• Comments
• first alignment - homo sapiens aldolase B gene
  that is
• readily available in
  the Genbank data base.
• - therefore, it has 100
  similarities in gene
• sequence.
• Both O.aries and Mus musculus aldolase 2 - 90
  similarities
• in gene sequence to the human aldolase B
  gene.
• Both Rattus norvegicus aldolase B(Aldob) and rat
  liver
• aldolase B - 88 similarities in gene
  sequence to the
• human aldolase B gene.

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b) Multiple DNA Sequence Alignment

• capable of comparing aldolase B gene with
  several other
• genes simultaneously.
• using CLUSTALW program
• - http//www2.ebi.ac.uk/clustalw/
• four different gene sequences(O.aries aldolase
  B, Mus
• musculus aldolase B, Rattus norvegicus
  aldolase B and rat
• liver aldolase B) are selected to compare with
  homo sapiens
• aldolase B for similarity.
• asterisk () all four subject genes have total
  similarity with
• aldolase B gene.
• part of the results are as below

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The tree view among the genes being compared with
aldolase B gene, it shows the distance of every
compared gene with the ancestor gene.
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STEP 6 Design primer for Polymerase Chain
Reaction(PCR) amplification

• Obtained liver biopsy tissue extracted mRNA of
  aldolase B gene
• Reverse transcriptase synthesized cDNA
• enzyme Klenow Fragment DNA polymerase -
  synthesize the second strand
• double-stranded cDNA for the following step PCR
  amplification.

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PCR amplification

• PCR - method for oligonucleotide primer directed
  enzymatic amplification of a specific DNA
  sequence of interest.
• forward primer and reverse primer needed
• Primer - short nucleic acid containing free
• 3 hydroxyl group
• - forms base pairs with a
• complementary template strand
• - functions as the starting point
  for
• addition of nucleotides to copy
• the template.

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• Forward primer 5 atg gcc cac cga ttt cca gc
  3
• Reverse primer 5 cta gta ggt ata gca ggc tg
  3
• directional cloning
• forward and reverse primer were prolonged -
  create restriction sites of EcoRI(gaattc) and
  HindIII(aagctt)
• both enzymes do not cut our gene of interest.
• atcgaattc (EcoRI) - 5' end of the forward primer
• aagcttatc (HindIII) - 5' end of the reverse
  primer
• atc bases served as extra sequences to stabilize
  the binding of the restriction enzymes.

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Polymerase chain reaction
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PCR procedure

• (a) Template denaturation
• - heating(95-97ºC), 15-30 seconds
• - denatures the template DNA
• (b) Primer annealing
• Tm (AT)2 (GC)4
• Forward primer (44)2 (48)4
• 64ºC - 2ºC
• 62ºC
• Reverse primer (55)2 (73)4
• 60 ºC - 2ºC
• 58 ºC
• Lowest temperature of the primers will be
• used 58ºC

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• GC content()
• - Forward primer 12/20 x 100 60
• - Reverse primer 10/20 x 100 50
• (C) Primer extension
• - DNA Polymerase is used to replicate the
• DNA segments
• - 70-72ºC, 15 minutes
• - procedure repeated many times
• - PCR product ALDOB gene

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STEP 7 Choosing a suitable vector for cloning

• Vector pETBlue-2 (Novagen catalog)
• special characteristics
• - presence of EcoRI and HindIII enzyme
  cleavage
• sites in the MCS
• - MCS - situated downstream of T7 promoter
• promotes high level protein expression in
  BL21
• E.coli host cells
• - presence of His Tag sequences and HSV Tag
  at the
• C-terminal - simplify and facilitate rapid
  protein
• purification and detection.

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• - Enterokinase cleavage site efficient
• removal of fusion tag.
• - Ampicillin resistance marker for
• screening.
• - high copy number pUC origin of
• replication increases plasmid yields
• relative to the vector and provides an
• advantage for sequencing and mutagenesis.
• - f1 origin of replication - allows
  single-stranded vector to be produced when
  co-infected with M13 helper phage.

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Plasmid map of pETBlue-2 vector.
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- pETBlue-2 digested by EcoRI and HindIII at
their respective cleavage sites located in the
MCS produced sticky ends at both ends.-
ensures directional cloning when the aldolase
B gene(contains EcoRI and HindIII restriction
sites at both 5 and 3 ends) is inserted and
ligated with the vector.
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STEP 8 Ligation

• Ligation - process where a DNA fragment, with
  blunt or sticky end, is inserted into an
  appropriate plasmid.
• DNA ligation involves creating a phosphodiester
  bond between the 3' hydroxyl of one nucleotide
  and the 5' phosphate of another.
• pETBlue-2 vector treated with shrimp alkaline
  phosphatase(SAP) - prevent self-ligation of the
  vector.
• T4 DNA ligase - ligate the ALDOB gene into the
  restricted pETBlue-2 vector.

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• pETBlue-2 with ALDOB gene insert

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STEP 9 Transformation into BL21 E.coli host

• BL21 E.coli is selected as the host cells
• Transformation - process by which extra-cellular
  DNA, usually plasmids isolated from cells, is
  taken in by a bacterial cell.
• Bacteria cells must be in a particular
  physiological state known as competence.
• Calcium chloride method
• - suspended bacteria in Cacl2
• - incubate on ice for 2 hour

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Calcium Chloride Method
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STEP 10 Verification Of Successful
Transformation

• to determine whether BL21 E. coli uptake the
  recombinant plasmid into the cell.
• culture BL21 E.coli on LB ampicillin agar plate
• colonies present on agar plate - transformation
  is successful.
• E.coli - sensitive to ampicillin in nature
• - will not grow on ampicillin
  plate.
• bacterial colonies observed on the plate
  conferred antibiotic
• resistance - had taken up the recombinant
  plasmid.
• transformed E.coli cells
• - protected by the ampicillin-resistance
  gene(AMP R) on the
• pETBlue-2 plasmid
• - express b-lactamase to inactivate the
  antibiotic ampicillin.

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STEP 11 Screening by using hybridization with
radioactive labeled probe

• colonies of BL21 E.coli that contain cloned
  segment of ALDOB gene - transferred from the
  culture medium onto a filter paper
  nitrocellulose(NC) filter.
• NC filter - treated with sodium hydroxide(NaOH)
  breaks open the cells and denatures the duplex
  DNA.
• NC filter - saturated with radioactive labeled
  probe complementary in base sequence to the ALDOB
  gene DNA strands are allowed to renature.

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• washing - remove unbound probe.
• positions of the bound probe identify the
  desired
• colony.
• detection of the desired colony - expose the
• nitrocellulose filter to X-ray film
  (autoradiography).
• orienting the film with original agar plate -
  colonies of
• E.coli carrying the recombinant DNA had been
• identified - the desired clone had been
  isolated.
•  
•  

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STEP 12 Expression of recombinant
protein

• isolated recombinant BL21 E.coli was grown on the
• nutrient agar plate.
• bacteria treated with IPTG
• IPTG
• - acts as an inducers to initiate the
  transcription
• and translation of the ALDOB gene into the
• aldolase B enzymes
• - leads to accumulation of large quantities
  of
• enzymes within the host cell.

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STEP 13 Extraction of protein

• previous step - provided us the unlimited
• amount of E.coli containing abundant
• human aldolase B enzymes.
• sonication method - lyse the bacterial
• cells to release the recombinant protein.

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STEP 14 Purification of recombinant
protein

• Nickel Metal Affinity Chromatography
• - pETBlue-2 contains His Tag sequence(high
• affinity for metals like nickel)
• - protein(human aldolase B enzyme) expressed
• in such vector will be a fusion protein
• - pass the recombinant protein with His Tag
• sequence through a Nickel affinity column.
• - His Tag sequence will bind to divalent
• cations (Ni2) immobilized on nitriloacetic
• acid (NTA) resins.

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- wash the unbound proteins or proteins without
the His Tag with buffer. - the target protein
is recovered by elution with either imidazole
or slight reduction in pH. - His Tag protein is
treated with enterokinase to cleave off the His
Tag. - pass the cleaved protein through the
Nickel affinity column once again to get the
recombinant protein freed of His Tag peptide.
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STEP 15 SDS-PAGE

• proteins are unravelled by boiling - disulfide
  bonds are
• broken with beta-mercaptoethanol.
• add detergent sodium dodecyl sulfate(SDS)
  linearises
• the protein chains and coats the protein
  with negative
• charge.
• migrate with the same mobility in stacking gel.
• begin to separate via molecular weight - sieving
  effect of
• the separating gel.
• separated proteins - visualized using staining
  method
• with Coomassie Brilliant blue R250.
• destained - observe the protein band.
• should be only one band - indicating molecular
  weight
• 40kDa approximately.

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Hunt for gene
ORF Analysis
Restriction enzyme analysis
Determination of amino acid sequence and
molecular weight
Compare gene sequence
PCR Amplification
Choosing suitable vector
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Ligation
Transformation
Verification of successful transformation
Hybridization with radioactive labeled probe
Expression of recombinant protein
Extraction of protein
Protein purification
SDS-PAGE
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USES AND COMMERCIAL VALUES OF THE HUMAN ALDOLASE
B ENZYME

• extensive study and thorough research
• - crystallographic structure of human
  aldolase B
• - mechanisms of control of Aldolase B
  expression
• - why HFI patients do not have difficulty
  maintaining their
• blood-glucose levels (in absence of
  fructose).
• - 2 hypotheses have been proposed
• (a) residual activity in mutant aldolase
  B
• (b) expression of the other aldolase
  isozymes.
• Both hypotheses - uncertain.
• - understanding the molecular basis of HFI
  - causes,
• distribution, biochemical and
  physiological effects.

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- determination of the biochemical roots of
this disorder - structural and
functional analyses will determine if the
HFI enzymes have any residual activity. -
enzymology of normal aldolase B Hopefully, a
cure for HFI disease will be discovered.
produce normal aldolase B enzyme in abundant
amount -produce relevant drug for the HFI
patients. introduced to HFI patients - direct
injection.
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CONCLUSION

• better understanding about the concept and
  principle of molecular biology techniques.
• gained precious knowledge and experiences
  regarding the procedures of molecular cloning.
• open our mind to genetic engineering field.
• had learnt a lot of things regarding the HFI
  disease.

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The End