MP942: Ligation and Expression of GST-Fused pGEX-KGSP Plasmid

This practical assignment details the ligation and expression of a GST-fused pGEX-KGSP plasmid, a modified E. coli protein expression vector, used for recombinant production and purification of glutathione-S-transferase (GST) fusion proteins. The experiment involved cloning Leishmania DNA inserts, encoding phosphorylation sites, into the pGEX-KGSP plasmid. The student performed restriction digests, agarose gel electrophoresis, and DNA sequencing to confirm the successful ligation of the gene of interest. Furthermore, recombinant expression of the GST-peptide fusion proteins in E. coli was performed, followed by SDS-PAGE and immunoblotting using anti-GST-HRP antibodies to confirm the presence of the recombinant proteins. The results, including graphical maps, sequence alignments, and gel images, demonstrated the successful ligation and expression of the Leishmania gene within the pGEX-KGSP vector, validating the use of this method for protein expression studies. The assignment also includes a discussion of the experimental procedures and results.

Running head: LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID
LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID
Name of the Student:
Name of the University:
Author Note:

1LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID
Introduction
Expression vectors are widely used as potent molecular tool for obtaining protein
expression in E. Coli cells. pGEX is an expression vector fused with GST coding sequence,
primarily used for ligating gene of interest with the plasmid and screening with glutathione S-
transferase expression. It also consists of Ptac promoter, repressor of lacI, ampicillin
resistance gene (ampR), and multiple cloning site (MCS). Anti-GST-HRP antibodies are
utilized for screening GST expression in cells, confirming successful ligation of the gene of
interest. A leishmania protein is digested with HindIII and EcoR1 to ligate with pGEX-KGSP
plasmid, and immunoblotting was performed followed by SDS-PAGE. Results inferred
successful ligation and expression of gene of interest. Graphical map of pGEX-KGSP
plasmid with circular topology was obtained via Serial Cloner software. Virtual translation of
the ligated expression vector was performed by using Expasy Translate tool. Immunoblotting
was executed using antibody against GST, to confirm successful ligation of the plasmid and
desired protein expression.

2LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID
Results
Figure 1:

3LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID
Figure: Restriction sites of pGEX-KGSP plasmid.
Figure 2:
Sequence results for the plasmid as returned from the company-
CGACTGTCTTCCCGAACGCGCGAGGCAGATCGTCAGTCAGTCACGATGAATTAAGCTTTGGC
TCTATACTCATATGTAGGGCTTAATTCTCTTCTCTTAATGACTCGAATTCCACCACCACCAC
CGGAAATTCCCGATCCACGCGGAACCAGATCCGATTTTGGAGGATGGTCGCCACCACCAAAC
GTGGCTTGCCAGCCCTGCAAAGGCCATGCTATATACTTGCTGGATTTCAAGTACTTATCAAT
TTGTGGGATAGCTTCAATACGTTTTTTAAAACAAACTAATTTTGGGAACGCATCCAGGCACA
TTGGGTCCATGTATAAAACAACATCAAGAGCGTCATACAACATGAAGTCAGGATGGGTTACA
TGATCACCATTTAAATATGTTTTATGACATAAACGATCTTCGAACATTTTCAGCATTTCAGG
TAGCTTGCTAAGAAAATCAACTTTGAGAGTTTCAAAGTCTTTACTATATGCAATTCTCGAAA
CACCGTATCTAATATCCAAAACCGCTCCTTCAAGCATTGAAATCTCTGCACGCTCTTTTGGA
CAACCACCCAACATGTTGTGCTTGTCAGCTATATAACGTATGATGGCCATAGACTGTGTTAA
TTTAACATCACCATCAATATAATAAGGAAGATTGGGAAACTCCAAACCCAATTCAAACTTTT
TGTTTCGCCATTTATCACCTTCATCGCGCTCATACAAATGCTCTTCATATTTTTCTTCAAGA
TATTCCAAAAGAAGTCGAGTGGGTTGCACAAGGCCCTTAATTTTCCAATAACCTAGTATAGG
GGACATGA

4LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID
Figure 3:
Figure 3a: pGEX-KGSP with insert

5LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID
Figure 3b: pGEX-KGSP-Insert with EcoRV, ScaI, HindIII, EcoRI restriction sites
Figure 4:
Figure 4a: DNA marker used on the agarose gel

6LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID

7LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID
Figure 4b: Gel of plasmid digest. DNA marker in Lane A. ScaI digested product (Lane B)
and HindIII + EcoRV digested product (Lane C).
Figure 5:
5’AGCTTATCGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGAAGCTGTG
GTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTC
TGGATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTT
GACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGG
AAACAGTATTCATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGCAACCCACT
CGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGG
TGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATA
TTGATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCAC
AACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTT
GGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAAGTTG
ATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACA
TATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGT
TTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTA
TTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTG
CAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCGGATCTGGTTCCGCG
TGGATCGGGAATTTCCGGTGGTGGTGGTGGAATTCTAGACTCCATGGGTCGACTCGAGCTCA
AGCTTAATTCATCGTGACTGACTGACGATCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAA
CCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCA
GACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAG
TCACGTAGCGATAGCGGAGTGTATAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATT
TTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAA
ATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATG
AGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACA
TTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAG
AAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAA
CTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGAT
Lane A= DNA Marker
Lane B= ScaI digested product
Lane C= HindIII+EcoRV digested product

8LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID
GAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGC
AACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAA
AAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGA
TAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTT
TGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCC
ATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACT
ATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGG
ATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAA
TCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCC
CTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGAC
AGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCA
TATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCT
TTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACC
CCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTG
CAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCT
TTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGC
CGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATC
CTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACG
ATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCT
TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG
CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCG
CACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACC
TCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCC
AGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCC
TGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTC
GCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATG
CGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATAAATTCCGACACCATCGA
ATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGG
TGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACC
GTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGT
CGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCG
GCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAG
CGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGA
TCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTT
CCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGA
AGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGT
TAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTC
ACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTT
TCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACG
ATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGAT
ATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTCAACCAC
CATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTC
AGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACC
CTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGC
ACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTC
ACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGT

9LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID
GAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGGATTCACTGGCCGTC
GTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACA
TCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGT
TGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAA
AGCTGGCTGGAGTGCGATCTTCCTGAGGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGAT
GCACGGTTACGATGCGCCCATCTACACCAACGTAACCTATCCCATTACGGTCAATCCGCCGT
TTGTTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCACATTTAATGTTGATGAAAGCTGG
CTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTGGAATT3’
Figure 5a: Alignment of the pGEX-KGSP plasmid

10LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID
Figure 5b: Alignment of the pGEX-KGSP plasmid

11LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID
Sequence of the generated expression plasmid-
CTAGACTCCATGGGTCGACTCGAGCTCAagctTAATTCATCGTGACTGACTGACGATCTGCCTCGCGCGTTTCGGTGATG
ACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCC
CGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATAA
TTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAG
GTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATG
AGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTAT
TCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGT
TGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTT
CCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCG
CCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAA
GAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAG
GAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCAT
ACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTA
CTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCG
GCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGG
TAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGA
TAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCAT
TTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCA
CTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAA
CAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTC
AGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCC
TACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAA
GACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACC
TACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCC
GGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCG
GGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAAC
GCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGA
TAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGG
AAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATAAATTCCGACACCATC
GAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGT
AACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTT
CTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCG
GGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAA
ATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGG
TGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAA
GCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGA
AGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTT
CTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAA
GGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGT
TGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTCAACCACCATCAAACAGGATTTTCGCCTGCTGGGG
CAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGT
GAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCAC
GACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGC
TTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACC
ATGATTACGGATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGC
AGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGA
ATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAGGCC
GATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCTACACCAACGTAACCTATCCCATTAC
GGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCACATTTAATGTTGATGAAAGCTGGCTAC
AGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTGGAATTAGCTTATCGACTGCACGGTGCACCAATGCTTCTGGCGTC
AGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCACTCCCG
TTCTGGATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCG
GCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGTATTCATGTCCCCTATACTAGGTTATT
GGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAG
CGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGG
TGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAG
AGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGAC
TTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAAC
ATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCCAA
TGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAA
TCCAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCGGATCT
GGTTCCGCGTGGATCGGGAATTTCCGGTGGTGGTGGTGGaatt
Figure 5c: Small letters denote repetitive sequences and insert portion is highlighted.

12LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID
Figure 6:
L D S Met G R L E L K L N S S Stop L T D D L P R A F G
K T V V Met S P I L G Y W K I K G L V Q P T R L L L E Y
L E E K Y E E H L Y E R D E G D K W R N K K F E L G L E
F P N L P Y Y I D G D V K L T Q S Met A I I R Y I A D K
H N Met L G G C P K E R A E I S Met L E G A V L D I R Y
G V S R I A Y S K D F E T L K V D F L S K L P E Met L
K Met F E D R L C H K T Y L N G D H V T H P D F Met L Y
D A L D V V L Y Met D P Met C L D A F P K L V C F K K R
I E A I P Q I D K Y L K S S K Y I A W P L Q G W Q A T F
G G G D H P P K S D L V P R G S G I S G G G G G I
Figure: Protein sequence of the GST-peptide fusion protein. Fused glutathione S-transferase
part is highlighted in ‘yellow’. ORF (Open reading frame) is highlighted in pink.

13LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID
Figure 7:
Figure 7a: Size of marker bands with molecular mass for SDS-PAGE
Figure 7b: SDS-PAGE performed on the DNA marker, non-GST induced cell lysate and
GST-induced cell lysate, stained with Coomassie brilliant blue.
A= DNA Marker
B= lysate of non-induced cells
C= lysate of induced cells

14LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID
Figure 8:
Figure: Immunoblot performed with anti-GST-HRP antibody shows GST-tagged cellular
protein expression in lane B. Lane A consists of DNA marker, stained by Coomassie brilliant
blue.
Discussion
pGEX vector is extensively used for cloning suitable gene into bacterial cell, to understand
the expression of a specific protein by in-vitro methods. It is purposely built with GST
(glutathione S-transferase) coding sequence as a marker for successive plasmid expression.
Figure 1 mentions graphical map of the pGEX-KGSP vector construct generated via Serial
Cloner software.
Figure 2 shows the agency provided sequence of the Leishmania gene that was used as an
insert for the experiment.
Parent plasmid (pGEX-KGSP) cloned with Leishmania peptide as insert, was derived as
software generated ligation product (Figure 3a). The company provided sequence was
transformed into anti-parallel (reverse complementation) due to utilization of reverse
Lane A= DNA Marker
Lane B= lysate protein tagged
with anti-GST-HRP antibody

15LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID
oligonucleotide primer PGEX3՛ (CCGGGAGCTGCATGTGTCAGAGG) for sequencing.
Figure 3b, shows restriction cut sites for EcoRV, EcoR1, HindIII, Sca1.
Figure 4a shows DNA marker. Figure 4b shows electrophoretic migration of DNA marker in
lane A, ScaI digested product in lane B and HindIII+EcoRV digested product in lane C.
Figure 5a demonstrates the alignment of the pGEX-KGSP plasmid, along with the linear map
of the sequence in Figure 5b. Sequence of the expression vector was highlighted with the
insert (Figure 5c).
Expasy generated amino acid sequence of the expression was shown in Figure 6, along with
the GST region highlighted.
DNA markers used for SDS-PAGE is listed in Figure 7a. Migration of DNA marker is shown
in lane A, GST non-induced cell lysate is shown in lane B and GST induced cell lysate is
shown in lane C, stained with Coomassie brilliant blue (Figure 7b).
Immunoblotting was performed using anti-GST-HRP antibody. Figure 8 shows migration of
DNA marker in lane A (blue color due to covalent bonding between DNA marker and
Coomassie brilliant blue) and lane B shows expression of GST-fused protein in cell lysate.