School Papers

INTRODUCTION In addition to this structure each monomer

INTRODUCTION

Macrophage Migration Inhibitory Factor (MIF) is a proinflammatory cytokine which had been described in 1966 as a molecule which inhibits the migration of macrophages thus giving the synonym to its name(Bach, Rinn, Meyer, Dodel, & Bacher, 2008). Depending on from which site it is released, MIF plays different roles like the stress-induced hormone, cytokine, tumor promoter, tumor suppressor, redox modulator, pro-angiogenic factor etc. Since it is released from various cells, especially from immune cells and plays certain roles in inflammation. Once MIF releases, it will induce the expression of proinflammatory mediators by macrophages and activates T cells, and will promote inflammation and immune response. MIF has an activity to counter regulates the immunosuppressive effects of glucocorticoids, and it promotes TNF-a and IL-1b production, leads to further MIF release and that increases the expression of cytokines, matrix-degrading enzymes, and cyclooxygenases.

MIF is relatively a small protein of around 12.5kDa. The primary structure of it possess 115 amino acids(Pantouris et al., 2015). This unique hormone is released by the pituitary gland and forms a trimer of identical subunits. Its monomers possess two anti-parallel ?-helices that pack against a four stranded ?-sheet. In addition to this structure each monomer has two ?-strands that interact with (Sun, Bernhagentt, Bucalat, & Lolis, 1996)?-sheets of adjacent subunits to form an interface between monomers. Thus, the three, ?-sheets forms a barrel containing a solvent-accessible channel that runs through the centre of the protein along a molecular 3-fold axis. Electrostatic potential maps shown that the channel has a positive potential, revealing that it binds to negatively charged molecules. The elucidated structure for MIF is unique among other cytokines or hormonal mediators(Sun et al., 1996).

 

Figure 1: Structure of MIF (Solvent-permeable Cavities)

(http://www.rcsb.org/pdb/home/home.do)

MIF possesses two motifs with catalytic activity, one of which has tautomerase and the other is involved in oxidoreductase activity(Huang et al., 2002). Tautomerase is especially involved in the conversion of D-dopachrome (a non-physiological substrate) to DHICA. In 1990s a work by Rosengren and colleagues revealed that in addition to this catalytic activity MIF also involved in the tautomerization of phenyl pyruvate substrate(Rosengren et al., 1996). Further proceedings disclosed that the active site of it is located in an inter sub-unit pocket on the MIF trimer.

Figure 2: Active site of MIF

Also for the successful action of MIF requires a proline residue at the N-terminus which act as a catalytic base(Lubetsky, Swope, Dealwis, Blake, & Lolis, 1999)(Stamps, Fitzgerald, & Whitman, 1998); other important amino acid residues are Lys-32, Ile-64, Tyr-95, and Asn-97 and interestingly all most all of them  are conserved(Robert Kleemann et al., 2000). Other than the tautomerase action MIF has  thiol protein oxidoreductase activity (TPOR)(R Kleemann et al., 1998). This activity is totally dependent on a CXXC motif around the cysteine residues at the positions 56 and 59, with Cys-59 being crucial as well as important for the activity. Even though this protein has a cysteine residue at 80 in its primary structure, that does not have a functional role in catalytic activity(Robert Kleemann, Kapurniotu, Mischke, Held, & Bernhagen, 1999). Very recently it is found that the protein can reversibly interconvert between reduced and oxidized states using antibodies that target the CXXC motif; these two forms are immunologically different from each other and are differentially elevated during different conditions(Thiele et al., 2015).

 

 

Figure 3:Biological functions of MIF

Investigations reveal that MIF is one of an important target for the drug discovery, so that it can be useful for the treatment of certain inflammations and cancers(Cheng & Al-Abed, 2006). Tautomerase is an appealing target for the development of drug and indeed the various actions of this protein can be influenced by the small-molecule inhibitors(Al-Abed & VanPatten, 2011).Likewise oxidoreductase also. There are certain drugs like Ebselen, an allosteric inhibitor of MIF, is found to interact with the three cysteines; modify and prevent them from trimer formation, although it could still inhibit MIF activities in a bis-serine MIF variant. p425, an another allosteric inhibitor of MIF, also found to inhibit the oligomer formation to an extent(Bloom, Sun, & Al-Abed, 2016)(Ouertatani-Sakouhi et al., 2010) Inactivating MIF is found to delay the progression of tumour development. ISO-1 and its analogue ISO-66 is also found to inhibit the activities of MIF. Taking this, Dr. Senapati’s lab is highly interested to find a novel potent, high star inhibitor of MIF so that it can be effective against various diseased conditions in which MIF is found to be a culprit.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

OBJECTIVES & PLANS

In my work, my goal is to express and purify recombinant Human MIF (hMIF-HA).

The objectives are:

1)     Cloning of human MIF (hMIF) in a prokaryotic expression vector (PET-22).

2)     Expression and purification of hMIF-HA.

For achieving my goals my plans are:

Ø Amplification and purification of MIF gene from human pancreatic stellate cells c-DNA

Ø Cloning of MIF into pET 22b vector

Ø Transformation of vector construct with MIF gene insert into DH5-? (E.coli competent cell)

Ø Colony PCR to check the presence of MIF gene in the plasmids isolated from DH5- ? strain.

Ø Sequencing of plasmids isolated from DH5- ?.

Ø Transformation of PET22b-MIF construct into BL-21 strain for MIF protein expression.

Ø Induction of BL-21 cells with IPTG and preparation of whole cell lysate.

Ø Native PAGE for checking the protein expression.

 

 

 

 

 

MATERIALS & METHODS

Ø Amplification of MIF gene from MIAPaCa c-DNA:

The cDNA use for the experiment was MIAPaCa c-DNA (available in Dr. Senapati’s lab). The primers used for this study were designed in the laboratory itself  using two restriction sites, NdeI and XhoI. The master mix was prepared of about 25µl. The master mix contains:

Components

Volume(µl)

Buffer

2.5

dNTPs

0.5

MgCl2

0.75

Primer

1
(Forward & Reverse)

cDNA

2

Taq Polymerase

0.25

Total

25µl
 

Figure 4:Components of PCR Master Mix

The reaction conditions and temperature used for PCR involves:

Reaction

Temperature (?)

Time

Lid Temperature

105

90

Initial Denaturation

95

2 min

Denaturation

95

45sec

Annealing

52

45sec

Primer Extension

72

1min

Repeated 30 Cycles

Final Extension

72

10min

Figure 5:PCR Reaction conditions and Temperatures

Ø Visualisation of PCR product in agarose gel electrophoresis

Agarose gel with a concentration of 1% was prepared for the visualization of PCR product in 1X TAE. The required amount of agarose was weighed and dissolved in the solvent by boiling. Ethidium bromide (0.5 ?g/ml) was added to the solution after boiling and poured it to the gel casting tray and left to solidify. The gel was placed in gel tank containing 1 × TAE buffer and the samples containing 6 × loading dye were loaded. The gel was viewed on a UV transilluminator and image captured with the help of Geldoc.

Components of 1X TAE (pH:8.3)

40mM Tris              :

2omM Acetate        :

1mM EDTA            :

Distilled Water       :

 

Components of 6X Loading Dye

EDTA                     :0.1M

Xylene cynol FF     :0.25%

Bromophenol Blue :0.25%

Glycerol                  :50%

Ø Purification of MIF gene

 The human MIF gene fragments were eluted from agarose gel after the DNA fragment had resolved sufficiently. The gel slice was excised out, placed in a microcentrifuge tube, and heated at 65 ºC till the gel piece melted. This was followed by purification of MIF gene by use of illustration GFXTMPCR and gel band purification kit according to manufacturer’s protocol.

Ø Vector selection

pET22b(+) vector was used for the cloning purpose as it carries an N-terminal pelB signal sequence for potential periplasmic localization in addition to C-terminal His•Tag® sequence. The sequence is numbered by the pBR322 convention. In addition to multiple cloning site, the vector itself contain Ampicillin resistant gene as a selectable marker so that the downstream selection of clones made easy.

 

Figure 6:pET22 Vector Map

Ø Restriction digestion of MIF gene and PET 22b vector

     The PCR product of humMIF and PET 22b vector were digested with NdeI and XhoI using following protocol:

The Master mix were prepared separately for humMIF and pET22(b). The digestion mixture was kept undisturbed at 37ºC for 3hrs in a water bath.

The reaction mix involves:

DNA                :20µl

Buffer              :5µl

Xho1               :2µl

Nde1               :2µl

Dis.water         :21µl

Total               :50µl

Ø Purification of restriction digestion products

 The digested products were subjected agarose gel electrophoresis. After their running on the gel purification was carried out by using illustration TM GFXTM PCR and gel band purification kit as described previously.

Ø Ligation of MIF gene and plasmid

 The desired digested products were subjected for ligation. The reaction mixture was kept at 16? for overnight.

The reaction mix contains:

Vector             :10µl

Insert               :5µl

10X Buffer      :2µl

Enzyme           :1µl

Dis.water         :2µl

Total            :20µl

Ø Transformation of MIF insert plasmid into cloning strain

The ligated product was transformed into DH5-? E. coli competent cell strain. 3µl of PET22b plasmid and 60µl of DH5- ? was taken in an Eppendorf. After the addition of plasmid to the competent cells the tube was kept on ice for about 30 minutes. Then the cells were subjected to heat shock at 42? for 90 seconds. Again, it is kept on ice for 5 minutes. Then750 ml of Luria Bertani broth was added to the tube and incubated at 37?for 1 hour. After incubation, centrifugation was done at 6000rpm for 3minutes to pellet down the cells. 800 µl of supernatant was discarded and the pellet was suspended in remaining supernatant. The cells were spread on Luria Bertani agar plates containing ampicillin and incubated at 37? for overnight to obtain the transformed cells.

Ø Colony PCR

 Colony PCR was carried out with the “so called”transformed colonies which grew on the LB-Ampicillin plate, to confirm the presence of perfect transformation. PCR master mix were prepared. A loopful of colony was inoculated in to the PCR master mix containing MIF specific forward primer and vector specific reverse one (T7 reverse primer). Agarose gel electrophoresis was carried out to confirm the presence of MIF in the vector.

The PCR mix contains:

Water              :10µl (required for the inoculation of colony)

Template         :120µl

dNTPs             :5µl

Buffer              :25µl

Enzyme           :2.5µl

Primers            :2µl

Water              :93.5µl

Total               :250µl

Reaction

Temperature
(?)

Time

Lid Temperature

105

90sec

Initial Denaturation

95

5min

Denaturation

95

45sec

Annealing

60

45sec

Primer Extension

72

1.30min

Repeated for 30 Cycles

Final Extension

72

5min

Figure 7:Colony PCR Conditions & Temperature

Ø Culture of transformed colonies and plasmid isolation

 The transformed colonies were inoculated in LB medium and kept for overnight incubation at 37?.  The culture obtained was subjected to centrifugation at 4500 for 10 minutes. The supernatant was discarded and the pellet containing lysed cells were used for isolation of plasmids. The plasmids were isolated using illustrationTM Plasmid Prep Midi Flow Kit.

Ø Sequencing of plasmids

 The ligated product was sequenced using Hum-MIF gene specific forward primer and PET22b specifc reverse primer through the sequencing facility available at ILS. The sequence obtained was aligned with the sequence of pET22b plasmid using snap gene software.

 

RESULTS AND DISCUSSION

 

 

 

Marker

 

Figure 8:Amplification of Hum MIF from MIAPaCa c-DNA

 

The MIAPaCa c-DNA were amplified using PCR. The image shows the amplification of MIF gene. The lane 1,2 and 3 depicts the MIF gene and lane 5 shows the marker. From this it is concluded that the size of MIF gene is around 300bp.

 

 

                                                      

Figure 9:Restriction Digestion of MIF and Vector