“CLONING AND EXPRESSION OF HUMAN β DEFENSIN 2 IN Escherichia coli ” Submitted By SEEMA ABRAHAM Reg. No: SJAHMGB006 2007-2009 Under the guidance of Dr. E. Sreekumar, Scientist C,Department of Molecular Virology, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram
INTRODUCTION OBJECTIVES MATERIALS METHODS RESULT AND DISCUSSION SUMMARY CONCLUSION SCOPE OF THE STUDY
DEFENSINS: Defensins are effector molecules of the innate host defense system with antimicrobial activity. The mammalian defensins can be subdivided into three main classes according to their structural differences: the α -defensins, β -defensins and θ -defensins. HUMAN β DEFENSIN 2: Human beta-defensin-2 (hBD2), first discovered in 1997 in human skin. hBD2 is a cysteine rich cationic antimicrobial peptide with low molecular weight. It is mainly produced by epithelial cells.
DNA sequence of the pET-28a-hBD2, which codes for the target peptide containing 49 amino acid with a molecular weight of 5.3 kDa. This includes the N (His) 6 Tag and the C-terminal 41 amino acid hBD2. MODE OF ACTION: HBD2 sequentially permeabilise the outer and inner membrane of E. coli . It form voltage dependent channels in membranes. It induce leakage of cytoplasmic content. FUNCTIONS: HBD2 kills bacteria and fungi on the surfaces of higher organisms. It also influence adaptive immunity by attracting immature dendritic cells.
APPLICATIONS: HBD-2 can be used in the treatment of chronic skin infections. It inhibits HIV replication in the intracellular environment. rec-hBD-2 can be used as an antigen for the generation of anti-hBD-2- monoclonal antibodies. STRAINS: E.coli JM109 is a convenient host for initial cloning of target DNA into pET vector and for maintaining plasmids. E.coli BL21 DE3 pLys S strain is convenient for expression of HBD2. It carries the gene for T7 RNA polymerase under lacUV5 control.
To clone the cDNA of Human β defensin -2 isolated from human blood in bacterial expression system (pET 28a+). To express the cloned gene in E. coli BL21 (DE3) pLysS.
Vectors - pGEM -T Easy, pET28a (+). Host - E.coli JM109, E.coli BL21 DE3 pLys S. Medium - LB Broth, LB Agar. Antibiotics - Kanamycin(25µg/ml),Ampicillin(50µg/ml). Enzymes - Taq DNA Polymerase, Nco I , Not I ,T4 DNA ligase. Antibodies - Monoclonal Anti-poly Histidine, mouse anti IgG HRP conjugate. Markers - 100bp ladder,1kb ladder,protein marker, Prestained marker.
Restriction digestion of pGEM-T- hBD2. Restriction Digestion of pET28a. DNA Purification of hBD2 and pET28a by GFX Gel- Band Purification System. Ligation of hbd2 and pET28 Transformation of E.coli JM109 with ligated pET - hBD2 Confirmation of Positive Clones by Colony PCR
Plasmid Isolation from Positive Clones by Alkaline Lysis Method. Sequence Confirmation of pET- hBD2 clone Transformation of Expression host (BL21 DE3 pLysS) with pET hBD2 SDS- PAGE Analysis Cell Disruption and Solubilization of Expressed Protein Western Blotting
Restriction Digestion of pGEM-T- hBD2 1 2 3 Lane 1:Undigested pGEM -T Easy – hBD2. Lane 2:Digested pGEM-T Easy – hBD2. Lane 3:100bp Marker. hBD2 fragment. Restriction digestion of hBD2 -pGEM-T Easy. Restriction digestion with the enzymes Nco I and Not I gave lower band ( hBD2 fragment) of size 160bp.The remaining upper bands were the backbone vector fragments.
Restriction Digestion of pET28a Lane 1: Undigested pET28a. 5239 bp 5 kb Lane 2: Digested pET28a. Lane 3: 1kb Marker . Restriction digestion of pET28a Digestion of pET28a with restriction enzymes NcoI and Not I gave a single band of size of 5239 bp.
DNA Purification of hBD2 and pET28a by GFX Gel-Band Purification System. hBD2 fragment pET28a fragment Lane 1: Purified hBD2 fragment. Lane 2: Purified pET28a fragment. Purified hBD2 and pET28 a fragments . The single band in each lane shows the purified fragments of hBD2 and pET28 a
Ligation of Nco I / Not I – hBD2 and Nco I / Not I pET28 Overnight ligation was performed at 4°C and the ligated product was used for transformation. Transformation of E.coli JM109 with Ligated pET - hBD2 Transformed colonies.
Confirmation of Positive Clones by Colony PCR Lane 1: Clone 1. Lane 2: Clone 2. Lane 3: Clone 3. 200 bp Lane 4: Clone 4. 160bp Lane 5: 100bpMarker Confirmation of Positive Clones by Colony PCR Four colonies were isolated and confirmed its molecular weight as160 bp.
Plasmid Isolation from Positive Clones by Alkaline Lysis Method. Lane 1: Plasmid. Plasmid Isolation of positive colonies.
Sequence Confirmation of pET- hBD2 clone Translated peptide sequence of the hBD2 – pET28 a clone and submitted sequence from the Genebank ( Accession NM_004942) which shows 100% similarity with HBD2 pET expressed peptide.
Transformation of Expression host (BL21 DE3 pLysS) with pET hBD2 Transformed colonies.
SDS- PAGE Analysis 6 kDa 5.3kDa Expression of hBD2 gene by induction with IPTG. Lane 1: BL21 DE3 pLys S, pET28a-hBD2 (0 hr Uninduced). Lane 2: BL21 DE3 pLys S, pET28a-hBD2 induction after 1 hour. Lane 3: BL21 DE3 pLys S, pET28a-hBD2 induction after 2 hour. Lane 4: BL21 DE3 pLys S, pET28a-hBD2 induction after 4 hour. Lane 5: BL21 DE3 pLys S, pET28a-hBD2 induction after 6 hour. Lane 6: Broad range protein marker. The induced band was visualized corresponding to the band below the 6 kDa protein of the polypeptide marker.
5.3 kDa Localization of the expressed protein in the cell. Lane 1:BL21 DE3 pLys S Mock. Lane 2: BL21 DE3 pLys S, pET28a-hBD2 (Induced supernatant). Lane 3: BL21 DE3 pLys S, pET28a-hBD2 induction after 6 hr (Induced pellet). Lane 4: Broad Range Protein Marker (NEB). The protein was found to be in the insoluble inclusion body, since the bands were found in the pellet loaded .
Western Blotting 1 2 3 4 5 5.3kDa 6 kDa SDS-PAGE and Western blotting analysis of hBD2. Lane 1: Uninduced Mock. Lane 1: Uninduced Mock. Lane 2: Uninduced. Lane 2: Uninduced. Lane 3: Induced – Insoluble fraction. Lane 3: Induced -Insoluble fraction. Lane 4: Induced – Soluble fraction. Lane 4: Induced -Soluble fraction. Lane 5: Protein Marker. Lane 5: Prestained Marker. The protein was released into the media after cell lysis and the concentration was high in the supernatant.
The restriction digestion of pGEM-T- hBD2 and pET28a was done. The fragments were purified and ligated and transformed E.coli JM109 were produced. The sequence confirmation was followed by the transformation of E.coli BL21 DE3 pLys S with pET- hBD2 . The IPTG induction was performed and its results were confirmed by SDS PAGE and Western blotting.
This study confirmed that the cDNA of human β defensin 2 was successfully cloned in E.coli JM 109. Also confirmed that the fusion protein was expressed in E.coli BL21 DE3 pLys S. The expressed protein was present in the inclusion bodies in the bacterial cells .
Recombinant-hBD-2 (rec-hBD-2) can be used as an antigen for the generation of anti-hBD-2-monoclonal antibodies. It is used for immunohistochemical research of human tumour. Also used for the research of intracellular targets of hBD-2. HBD-2 can be used in the treatment of chronic skin infections. Human β defensin -2 can be used as an ideal therapeutic agent avoiding the problems of acquired resistance.
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