SUMMER UNDERGRADUATE RESEARCH PROGRAM-2016

transcript

01/05/2023

SUMMER

UNDERGRADUATE

RESEARCH PROGRAM-

2016A C B R N E W

D E L H I

B Y S A U M Y A K U M A R

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EXPRESSION AND PURIFICATION OF MTRR PROTEIN OF NEISSERIA

GONORRHOEAE

NEISSERIA GONORRHOEAE

• Is an obligate gram negative, diplococcus, aerobic bacteria.

INTRODUCTION

• In absence of vaccine antibiotic therapy is recommended as treatment regimen on the basis of symptoms alone.

• Second most important STD causing pathogen.

• Large number of multidrug resistant isolates are reported in various studies across the world.

• An in depth understanding of gonococcal antibiotic resistance mechanism is essential for the development of new and effective antimicrobial agents.

MTRR NEGATIVE REGULATOR OF MTRCDE EFFLUX PUMP

MtrR is a tetR family member.Repressor of MtrCDE efflux pump. It has 53% identity and 78% homology with AcrR.Helix rich protein with HTH DNA binding domain and C’-terminal ligand binding domain.Binds to pseudo-direct invert repeat promoter region.Mutation in promoter region or in MtrR impart resistance to bacteria for various hydrophobic drugs.A/T deletion in 13bp invert alters the distance between -10 and -35 region of mtrR promoter and thus binding of MtrR to its promoter.

TECHNIQUES LEARNT

PCR SDS-PAGEWestern BlottingProtein Purification

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POLYMERASE CHAIN REACTION (PCR) AND ITS APPLICATIONS

WHAT IS PCR?

It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993.

PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro.

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WHY “POLYMERASE”?

It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.

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WHY “CHAIN”?

It is called “chain” because the products of the first reaction become substrates of the following one, and so on.

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The “Reaction” Components

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1) Target DNA - contains the sequence to be amplified

2) Pair of Primers - oligonucleotides that define the sequence to be amplified.

3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.4) Thermostable DNA Polymerase - enzyme that catalyses the reaction5) Buffer solution – maintains pH and ionic

strength of the reaction solution suitable for the activity of the enzyme.

Denature (heat to 95oC)

Lower temperature to 58oC Anneal with primers

Increase temperature to 72oC DNA polymerase +

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APPLICATIONS OF PCR• Classification of organisms

• Genotyping• Molecular archaeology

• Mutagenesis• Mutation detection

• Sequencing• Cancer research

• Detection of pathogens

• DNA fingerprinting

• Drug discovery• Genetic matching• Genetic engineering

• Pre-natal diagnosis

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RESULTL NC PC T

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L : DNA LadderNC : Negative ControlPC : Positive Control T : Test Sample

665 bp

265 bp

700 bp

300 bp

Sodium Dodecyl Sulphate- Polyacrylamide Gel Electrophoresis (SDS-PAGE)

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Electrophoresis is a laboratory technique for

separating molecules based on their charge.

What is Electrophoresis?

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SDS-PAGE• SDS-PAGE ( sodium dodecyl sulphate-

polyacrylamide gel electrophoresis)

• The purpose of this method is to separate proteins according to their size, and no other physical feature

• In order to understand how this works, we have to understand the two halves of the name: SDS and PAGE

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Sodium Dodecyl Sulphate

Now we are ready to focus on the second half - PAGE.

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Procedure• Prepare polyacrylamide gels.• Add diluted samples to the sample buffer.• Heat to 100C for 5 minutes.• Load the samples onto polyacrylamide gel.• Run at 70 volts for 3 hours.• Stain.• Destain.

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Gel PreparationReagents 10% Resolving

GelStacking Gel

dd H20 4.0 ml 3.4 ml

30% Acrylamide 3.3 ml 830 µl

1.5M Tris Base(pH=8.8) 2.5 ml --

1M Tris Base(pH=6.8) -- 625 µl

10% SDS 100 µl 50 µl

10% Ammonium Persulphate

100 µl 50 µl

TEMED 10 µl 5.0 µl

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Running Buffer• Tris buffer to provide appropriate pH.

• SDS (sodium dodecyl sulphate) detergent to dissolve proteins and give them a negative charge.

• Glycine different charge properties in different pH. In stacking gel(pH=6.8) it is Uncharged and in resolving gel(pH=8.8) it is negatively charged.

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Result

UI I UI I UI I UI I UI I L

In the Given figureUI : Un Induced Protein I : Induced Protein L : Protein Ladder

Western Blotting

Western blotting, also known as immunoblotting or protein blotting, is a technique used to detect the presence of a specific protein in a protein mixture

Steps involved in western blotting

1. Sample preparation2. Gel Electrophoresis3. Blotting (or transfer)4. Blocking5. Antibody Probing6. Detection

Sample Preparation• All sources of protein, from single cells to whole

tissues, biological fluids and proteins secreted in vitro, are open to analysis by Western blotting.

• In most cases, the cells are harvested, washed, and lysed to release the target protein.

• For best results, all these steps should be carried out on ice.

Gel Electrophoresis- Gel Preparation• Mix ingredients in the order shown above,

ensuring no air bubbles form.• Pour the separating gel into glass plate

assembly • Overlay gel with water to ensure a flat surface

and to exclude air.• Leave to polymerize for ≃20 minutes.• Then prepare the stacking gel and pour on the

running gel, insert comb and leave for 20 min.

Blotting• The membrane is placed next to the gel.• The two are sandwiched between absorbent

materials, and the sandwich is clamped between solid supports to maintain tight contact between the gel and membrane.

• The system is connected to a power supply and blotting is run for 40 min at 70 V.

Blotting Membranes• The solid support onto which the separated

proteins are transferred is usually of two types, both of which bind proteins with high affinity:A. Nitrocellulose membrane

• has excellent protein binding and retention capabilities

• is brittle and thus it is usually less effective when blots need to be reused.

B. Polyvinylidene fluoride (PVDF) membrane• PVDF demonstrates superior mechanical

strength making it suitable for stripping/reprobing.

Blocking• Blocking is a very important step in the

immunodetection phase of Western blotting because it prevents non-specific binding of antibody to the blotting membrane

• The most commonly used blocking solutions contain 3-5% BSA in a solution of PBS (phosphate buffered saline).

• Often, a small amount of Tween 20 detergent is added to blocking and washing solutions to reduce background staining, and the buffer is known as PBST.

Antibody Probing• The protein of interest is detected and localized

using a specific antibody.• The blot will be incubated in a dilute solution of

antibody, usually for a few hours at room temperature or overnight at 4°C.

• The antibody is diluted in wash buffer (PBST) or in the blocking solution, the choice depends upon the antibody.

• Since antibody preparations vary in their levels of purity and specific binding properties, there will be differences in the level of dilution required.

Antibody Probing• After incubation with Primary Antibody ash the

membrane several times in PBST while agitating, 15 minutes or more per wash, to remove residual primary antibody.

• A species-specific, labeled secondary antibody directed against the constant region of the primary antibody is then used.

• The secondary antibody serves not only as a carrier of the label but is also a mechanism to amplify the emitted signals, as many secondary antibodies can theoretically bind simultaneously to the primary antibody.

Results M UI E 1 E 2

M : Protein MarkerUI : Uninduced Protein E : Elution

Protein Purification

Ni-NTA (Nitrilo Tri Acetic acid) Affinity Chromatography

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Solutions • Charging Buffer : Contains NiSO4 to charge the Beads

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• Binding Buffer : makes possible the binding of Protein to the Beads

• Wash Buffer : To wash that is not or very weekly bound.

• Elution Buffer : To elute the Protein from Beads.

Affinity Chromatography

• To purify our protein from other bacterial proteins.

• The column contains a compound of Ni that helps purify the protein. • Histidine in the protein forms a physical bond with Ni, thus binding the column.

Bind denatured protein to the Ni-Agarose column on the shaker for at least one hour.

Only our sensor domain with a 6X His tag would have bound the column strongly.Elute with the elution buffer with little amount of imidazole in it, and collect fractions.

Wash it with the wash buffer to remove:• anything that did not bind the column, and• anything that bound very weakly.

Affinity Chromatography• Elute the column with elution buffer, increasing the amount of imidazole in it each time.• Imidazole replaces His on the column and the protein is released.

• Run samples from the fractions on the gel and pool together the ones that have only our protein in it.

• All the protein on the column would come out, and be collected into fractions.

o ug h

35 kD28 kD

Thank you

SUMMER UNDERGRADUATE RESEARCH PROGRAM-2016

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