Expt.

1: ESTIMATION OF PROTEIN CONCENTRATION

OBJECTIVE: To estimate the protein concentration using Bradford method and Lowry method

PRINCIPLE:

Bradford assay: It is the protein determination method that involves the binding of the dye
Coomassie Brilliant Blue G-250 to proteins. The dye exists in three forms: cationic (red), neutral
(green) and anionic (blue). Under acidic conditions the dye is predominantly in the doubly
protonated red cationic form (Amax=476nm). However when the dye binds to the protein, it is
converted to a stable un-protonated blue form (Amax=595nm). The basic mechanism of the
assay is the binding of dye at acidic pH to Arginine, Histidine, Phenylalanine, Tryptophan and
Tyrosine residues and hydrophobic interactions. Upon binding protein, a metachromatic shift
from 465 to 595 nm is observed due to stabilization of the anionic form of the dye. Certain
chemical- protein and chemical- dye interactions interfere with the assay. Interference from non-
protein compounds is due to their ability to shift the equilibrium levels of the dye among three
colored species- known source of interference, such as some detergents, flavonoids and basic
protein buffers, stabilize the green neutral dye species by directly binding or by shifting the pH.

Lowry assay: The phenolic group of Tyrosine and Tryptophan residues in a protein will produce
a blue- purple color complex, with maximum absorption in the region of 660nm wavelength,
with Folin- Ciocalteu reagent which consists of sodium tungstate/ molybdate and phosphate.
Thus the intensity of color depends on the amount of these aromatic amino acids present and will
thus vary for different proteins. Most protein estimation techniques use BSA because of its low
cost, high purity and availability. The method is sensitive down to above 10g/ml, if subjected to
interference from this buffer, EDTA, anionic and cationic detergents, carbohydrates, lipids and
some salts. The incubation time is very critical for a reproducible assay. The reaction is also
dependent on pH and a working range of pH 9 to 10.5 is essential.

REAGENTS REQUIRED:

Bradford assay: Dissolve 100mg Coomassie Brilliant Blue G-250 in 50ml of 95% ethanol and
add 100ml 85% phosphoric acid while stirring continuously. When the dye has dissolved, dilute
to 1 litre in water. The reagent is stable for upto a month at room temperature. Filter the reagent
using Wattman’s filter paper no.1 and store the reagent in amber colored bottle.

Lowry reagent:

Reagent A: sodium potassium tartrate – 7mM, sodium carbonate (0.81 M), 0.5M NaOH

Reagent B: 70mM sodium potassium tartrate, 40mM copper sulphate

Reagent C consists of 1 volume Folin- Ciocalteu reagent diluted with 15 volume water.
PROCEDURE:

Bradford Assay-

1. Prepare standards in the range of 100- 1500 μg/ml in a Bradford- compatible buffer. The
more dilute sample, the sensitivity can be increased by increasing the ratio of sample to
reagent volumes (1-25 μg/ml). if the ratio of sample to dye is too high, the pH of reaction
mixture could increase leading to higher background responses.
2. Add the standard and unknown samples to the cuvettes.
3. Allow Bradford reagent to warm to room temperature. Add 1ml of dye to 25μl of protein
sample, mix and incubate for 10 minutes at room temperature.
4. Measure the absorbance at 450 and 595 nm.
5. Plot either 595nm data or improved precision at lower response values the ratio 595nm/
490nm.
Standard curve can be fit to a polynomial response from which known protein estimate can
be calculated.

Lowry Assay-

1. Prepare a series of dilutions of 0.3mg/ml BSA in the same buffer containing the
unknown, to give concentration of 30 to 150 μg/ml (0.03 to 0.15 mg/ml).
2. Add 1ml each dilution of standard protein containing unknown or buffer to 0.9ml reagent
A in separate test tubes and mix.
3. Incubate the tubes for 10 min in 50 ̊C bath, then cool to room temperature.
4. Add 0.1ml reagent B to each tube, mix, incubate for 10 min at room temperature.
5. Rapidly add 3ml reagent C to each tube, mix, incubate for 10 min in 50 ̊C bath and cool
temperature. Final assay volume is 5ml.
6. Measure absorbance at 650nm in 1cm cuvettes.
Prepare standard curve of absorbance vs (μg) of protein and determine amounts from the
curve.

OBSERVATIONS:

BSA stock concentration = ……….. mg/ml

BSA Volume of stock Volume of water Bradford reagent OD

concentration (μl) (μl) (ml)
(μg)

RESULT:
Expt No.2 Separation Of Proteins And Estimation Of Molecular Weight By Sodium Dodecyl
Sulfate-Polyacrylamide Gel Electrophoresis

Aim: The objective of the experiment is to resolve the proteins on a SDS-PAGE and identify its
molecular weight

Introduction:
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) is an excellent method
to identify and monitor proteins during purification and to assess the homogeneity of purified
fractions. SDS-PAGE is routinely used for the estimation of protein subunit molecular weights
and for determining the subunit compositions of purified proteins. Most electrophoresis is done
in vertical chambers in gel slabs formed between two glass plates. The slab format provides
uniformity, so that different samples can be directly compared in the same gel. Gel thicknesses
are established by spacers placed between the glass plates and sample wells are formed in the
gels during polymerization with plastic, comb-shaped inserts. Conventional gels are of the order
of 16–20 cm long, 16 cm wide, and 0.5–3.0 mm in thickness and can accommodate about 25
samples. Thick gels have greater total protein capacity than thin ones, but are correspondingly
less efficient at dissipating electrically generated heat and more difficult to stain and destain.
Typical runs take 4–5 h.

Polyacrylamide gels are formed by copolymerization of acrylamide monomer, CH 2=CH--CO--

NH2, and a cross-linking comonomer, N,N '-methylenebisacrylamide, CH2=CH--CO--NH--
CH2---NH--CO--CH=CH2, (bisacrylamide). The mechanism of gel formation is vinyl addition
polymerization and is catalyzed by a free radical-generating system composed of ammonium
persulfate (the initiator) and an accelerator, tetramethylethylenediamine (TEMED). TEMED
causes the formation of free radicals from persulfate and these in turn catalyze polymerization.
Oxygen, a radical scavenger, interferes with polymerization, so that proper degassing to remove
dissolved oxygen from acrylamide solutions is crucial for reproducible gel formation. The
sieving properties of a gel are established by the three-dimensional network of fibers and pores
which is formed as the bifunctional bisacrylamide cross-links adjacent polyacrylamide chains.
Within limits, as the acrylamide concentration of a gel increases, its effective pore size
decreases. The effective pore size of a gel is operationally defined by its sieving properties; that
is, by the resistance it imparts to the migration of protein molecules.
Principle:
Principle of Electrophoresis: Under the influence of an electrical field charged molecules
and particles migrate in the direction of the electrode bearing the opposite charge. Because of
their varying charges and masses, different molecules and particles of a mixture will migrate at
different velocities and will thus be separated into single fractions. The movements of the
particles are retarded by interactions with the surrounding gel matrix, which acts as a molecular
sieve. The opposing interactions of the electrical force and molecular sieving result in differential
migration rates for the constituent proteins of a sample.
In general, fractionation by gel electrophoresis is based on the sizes, shapes, and net
charges of the macromolecules. Systems designed to fractionate proteins in their native
configurations cannot distinguish between the effects of size, shape, and charge on
electrophoretic mobility. As a consequence, proteins with differing molecular weights can have
the same mobility in these systems. Thus, while PAGE methods for native proteins are valuable
for separating and categorizing protein mixtures, they should not be used to assess the purity of a
preparation or the molecular weight of an unknown. SDS-PAGE overcomes the limitations of
native PAGE by imposing uniform hydrodynamic and charge characteristics on all the proteins
in a sample mixture. During sample preparation, proteins are treated with hot SDS. The anionic
detergent binds tightly to most proteins at about 1.4 mg of SDS/mg of protein, imparting a
negative charge to the resultant complexes. Interaction with SDS disrupts all noncovalent protein
bonds, causing the macromolecules to unfold. Concomitant treatment with a disulfide-reducing
agent, such as 2-mercaptoethanol or dithiothreitol, further denatures proteins, breaking them
down to their constituent subunits. The electrophoretic mobilities of the resultant detergent-
polypeptide complexes all assume the same functional relationship to their molecular weights.
Migration of SDS derivatives is toward the anode at rates inversely proportional to the
logarithms of their molecular weights. SDS polypeptides, thus, move through gels in a
predictable manner, with low-molecular-weight complexes migrating faster than larger ones.
This means that the molecular weight of a protein can be estimated from its relative mobility in a
calibrated SDS-PAGE gel and that a single band in such a gel is a criterion of purity.
Procedure:

1. Assemble the casting apparatus and determine the gel volume from the manufacturer's
instructions or by calculation.
2. A 1- to 2-cm stacking gel is used above the resolving gel. Determine the height to which
the resolving gel is to be poured by inserting a well-forming comb between the glass
plates and marking the outer plate 1-2 cm below the teeth of the comb.
3. Prepare the monomer solution for the appropriate resolving gel by combining all of the
reagents in Table except the ammonium persulfate (APS) and TEMED
4. Deaerate the solution under vacuum (e.g., in a bell jar or desiccator) for at least 15 min.
5. Gently mix the APS and TEMED (according to the concentration of the gel as per
theTable) into the deaerated monomer solution.
6. Using a pipet and bulb, add the monomer solution between the gel plates up to the mark
delimiting the resolving gel.
7. Immediately overlay the monomer solution with water-saturated 2-butanol or water.
8. Allow the gel to polymerize for 45 min to 1 hr.
9. Prepare stacking gel monomer solution as per the table.
10. Thoroughly rinse the top of the resolving gel with water and dry the area above it with
filter paper. Place a well-forming comb between the gel plates and pour the stacking
monomer solution mixed with the appropriate volume of APS and TEMED.
11. Allow the gel to polymerize for 30-45 min.
12. Sample Preparation: Dilute samples with at least 4 vol of complete SDS-reducing
buffer
13. Heat the diluted samples at 95 °C for 4 min by suspending the sample tubes in hot water.
1. Load the sample in the wells and start the electrophoresis power pack.
2. Run the gel at 8-10 V/cm until the bromophenol blue reaches the bottom of the stacking
gel and then increase the current to 12-18 V/cm until the bromophenol blue reaches
bottom of the resolving gel.
3. Stain the gel with Coomassie Brilliant Blue to visualize the induced protein.

Discussion and conclusions:

Buffers and reagents required for SDS- PAGE

Acrylamide (50 ml).

(Store at 4oc)

30 % Acrylamide.
0.8 % N, N Methylene Bis-acrylamide.
Steps:
1) Weigh 15 g acrylamide accurately and dissolve it in 20ml of distilled water.
2) Weigh 0.4 g bisacrylamide separately and add it in 20ml distilled water. Keep on stirrer for
30min at the minimum until it is completely dissolved.

3) Make up the volume to 50ml.

4) Acrylamide is light sensitive so store it in Dark bottle and also cover the beaker with foil
while it’s on stirrer.

10 % Ammonium per sulfate (APS)

(Stored at 4o c)

Weigh 0.1 g in 1 ml distilled water.

It needs to be prepared fresh always. Can be used for one week
(Provided it is stored at 4 oC).

10 % SDS (Sodium dodecylsulfate) or Lauryl Sulfate.

(Store at room temp.)

For 10 ml weigh 1 gm SDS, avoid frothing while preparing. Don’t vortex it , can leave it
overnight if it is not dissolved completely.
Stored at room temperature.

1.5 M Tris, pH 8.8 ( 50 ml) For Resolving or Separating gel.

(stored at 4oC)

Molecular weight:121.4
Weigh 9.08 g Tris and adjust the pH to 8.8 and make up the volume to 50 ml.

0.5 M Tris, pH 6.8 (50 ml) For Stacking gel.

(stored at 4oC)

Molecular wt: 121.4

Weigh 3.03 g Tris and adjust the pH to 6.8 and make up the volume to 50 ml.

Loading Dye (6 X): 10 ml. (stored at 4oc)

7 ml Tris-Hcl pH 6.8 (0.5 M)

1 g SDS.
3 ml Glycerol.
0.93 g DTT (1.5 % β-ME)
1.2 mg Bromophenol Blue.
Running Buffer (1 X)

For 500 ml For 300 ml

Glycine: 7.2 g 4.32 g
Tris :1.51g 0.906 g
SDS :0.5 g 0.3 g
(stored at 4oc)

Staining solution: (500ml)

(Stored at Room temp.)
0.2 g Coomaasie Blue
200 ml Methanol.
25 ml Acetic acid.
275 ml distilled water.
For 10 % Acrylamide gel
Resolving or Stacking gel (5%)
Total volume Separating gel (10 %)
8 ml 15 ml 30 mL 3 ml 4 ml 5 mL
H2O 3.2 5.9 11.8 1.72 2.2 2.77
30% Acryl mix 2.64 5.00 10.0 0.5 0.67 0.83
1.5 M Tris(pH 8.8) 2.0 3.8 7.6 ------ ------ ------
0.5 M Tris (pH6.8) ----- ------- ------ 0.76 1.0 1.26
10% SDS 80 µl 150 µl 300 µl 30 µl 40 µl 50 µl
10% APS 80 µl 150 µl 300 µl 30 µl 40 µl 50 µl
TEMED 3.2 µl 6 µl 12 µl 3.0 µl 4 µl 5 µl
 Expt: 3 Bacterial Cell Disruptions

Aim: To study and analyze the efficiency of bacterial cell disruption using either non-ionic detergents
like Triton X-100 or only lysozyme or only sonication.

Introduction:
Biotechnological products produced by different types of cells can be intracellular or
extracellular. If these are intracellular, the cells have to be disrupted to release these products before
further separation can take place. There are a variety of reliable methods for cellular disintegration and
extraction of proteins ranging from enzymatic digestion and osmotic shock to ultrasonication, and
pressure disruption. Each method has inherent advantages and disadvantages. Generally vigorous
mechanical treatments reduce extract viscosity but can result in the inactivation of labile proteins by heat
or oxidation, while gentle treatments may not release the target protein from the cells, and resulting
extracts are extremely viscous. Depending on the cell type selected as the source for target protein
expression, cellular extracts contain large amounts of nucleic acid, ribosomal material, lipids, dispersed
cell wall polysaccharide, carbohydrates, chitin, small molecules, and thousands of unwanted proteins.
Cell disruption techniques must rapidly and efficiently lyse cells to extract proteins with minimal
proteolysis or oxidation while reducing extract viscosity caused by cell debris and genomic DNA
contamination. Different cell disruption techniques that are commonly used include:
Physical methods
a) Disruption in ball mill or pebble mill
b) Disruption using a colloid mill
c) Disruption using French press
d) Disruption using ultrasonic vibrations
Chemical and enzymatic methods
a) Disruption using detergents
b) Disruption using enzymes e.g. lysozyme
c) Combination of detergent and enzyme
d) Disruption using solvents

Principle:
Micro-scale cell disruption is often accomplished through chemical and enzymatic methods or
combinations of the two. The active ingredients in many of the bacterial lysis reagents are nonionic or
zwitterionic detergents functioning to disrupt cell membrane and cell wall structures weakening the cells
for rupture by osmotic shock, freeze-thaw, or enzymatic attack by lysozyme. Gram positive bacteria are
easily disrupted by lysozyme treatment alone, with a few exceptions. Gram negative bacteria are difficult-
to-disrupt with lysozyme alone because their outer lipid bilayer must be permeabilized first to expose the
peptidoglycan cell wall to lysozyme digestion. Detergents break the lipid:lipid, protein:protein,
protein:lipid interactions. In general, non-ionic or zwitterioninc detergents are milder, resulting in less
protein denaturation upon cell lysis, than ionic detergent and are used to disrupt cells when it is critical to
maintain protein function or interactions. The non-ionic detergents like Triton X-100 and nonidet P-40 are
commonly used for the purpose. In contrast, ionic detergents are strong solubilizing agents and tend to
denature proteins, thereby destroying protein activity to function. Tris buffer plus EDTA liberates about
50% of the polyanionic lipopolysaccharide in the bilayer, but EDTA interferes with the most popular
downstream purification step for recombinant proteins, immobilized metal affinity chromatography
(IMAC). The detergent lysis reagents are extremely effective for outer membrane permeabilization
without IMAC interference. Apart from Tris, EDTA and detergents, the cell lysis buffer also contains,
glycerol (5-10%) which maintains the stability of the proteins, sodium chloride to provide osmotic shock,
Strong reducing agents like β-mercaptoethanol or Dithiothreitol (DTT) may be included in the lysis
to limit proteolysis and improve target protein solubility and stability.
 Enzymatic treatment of microbial cells for lysis and viscosity reduction has several distinct
advantages over mechanical and chemical methods. The lytic enzymes are highly specific for targeted cell
wall components, they are gentle and do not generate shear, high temperature or oxidative damage, and
they are simple to use requiring no specialized equipment for processing.
The physical methods of cell disruption like Sonication and high-pressure disruption have been
effectively applied for disruption of microorganisms, plants, and animal cells. Sonication is based on the
shear forces created by high frequency ultrasonic vibrations generated by resonation (15–25 kHz) of a
tuned probe or horn. The sonic pressure waves created cause the collapse of formed microbubbles and
their implosion generates shock waves with sufficient energy to disrupt cell walls, and reduce viscosity by
shearing nucleic acids.
The components and volume of cell disruption buffers are critical not only for efficient disruption
but will also affect subsequent purification steps and the target protein’s stability and recovery after it has
been released from the cells. Each protein extracted is an individual and ideally would have an extraction
and purification buffer tailored specifically for its own biochemical requirements and intended direction
through the purification pipeline. In most cases a more generic extraction buffer can be used with good
results if a few basic criteria are met. These criteria are pH, ionic strength, additives to prevent
degradation and improve stability, and buffer to cell paste ratio.

Procedure:
1. Inoculate 50 mL autoclaved LB media with the bacterial cells and incubate overnight in an orbital
shaker at 120 RPM.
2. Harvest the cells by centrifugation at 9000 rpm for 5 minutes in a centrifuge at 4oC
3. Remove and discard the spent medium by aspiration.
4. Resuspend the cell pellets in 2 mL of Phosphate buffered saline (PBS) and harvest the cells by
centrifugation as given in step 2
5. Resuspend the cell pellets in 500 µL of ice cold lysis buffer containing
(i) 0.1% Triton X-100 or
(ii) 1 mg/mL of lysozyme or
(iii) Nothing added to the lysis buffer
6. Mix the sample in (i) and (ii) by pipetting and incubate on ice for 90 hr
7. Vortex the cells every 10 minutes and incubate on ice.
8. Mix the sample (iii) by pipetting and disrupt the cells by ultrasonication with ten pulses of 30 s each
at 30 s intervals.
9. Centrifuge the lysates for 10 min at 9000 rpm at 4oC and separate the supernatant from the cell debris
10. Remove a 2-10 µL sample of the supernatant for analysis of soluble proteins by Bradford assay.

Discussion: …..

Buffers and reagents required for this experiment

LB Medium (Luria-Bertani Medium) (For 1 liter):

To 950 ml of deionized H2O, add:
Tryptone 10 g
Yeast extract 5 g
NaCl 10 g
Shake until the solutes have dissolved. Adjust the pH to 7.0 with 5 N NaOH (0.2 ml). Adjust the volume
of the solution to 1 liter with deionized H2O. Sterilize by autoclaving for 20 minutes at 15 psi on liquid
cycle.
Phosphate-buffered Saline (PBS)
137 mM NaCl,
2.7 mM KC1,
10 mM Na2HPO4,
2 mM KH2PO4,
Dissolve 8 g of NaC1, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4, in 800 ml of distilled
H2O. Adjust the pH to 7.4 with HCl. Add H2O to 1 liter. Dispense the solution into aliquots and sterilize
them by autoclaving for 20 minutes at 15 psi on liquid cycle or by filter sterilization. Store the buffer at
room temperature.
Lysis Buffer
Tris Hcl 50 mM, pH 8.0
NaCl 50 mM
Glycerol 5%
EDTA 0.5mM
β-mecaptoethanol 0.1% (To be added just before starting the experiment)
Dithiothreitol (DTT) 0.5 mM
Lysozyme (10 mg/ml)
Dissolve solid lysozyme at a concentration of 10 mg/ml in 10 mM Tris-C1 (pH 8.0) immediately before
use. Make sure that the pH of the Tris solution is 8.0 before dissolving the protein. Lysozyme will not
work efficiently if the pH of the solution is less than 8.0.

Expt: 4 Bacterial Cell Disruptions by combining multiple techniques

Aim: To study and analyze the efficiency of bacterial cell disruption using (A) Triton X-100 and
lysozyme and (B) sonication in the presence of Triton X-100 and lysozyme (C) sonication in the presence
of Triton X-100 and lysozyme after mechanical disruption in a homogenizer.

Principle:
Although enzymatic treatments give good results in combination with a non-ionic detergent for
cell disruption, enzyme treatments of cells and extracts are generally combined with mechanical
disruption methods.
Sonication and high-pressure disruption are the mechanical methods that are widely used in combination
with the enzymatic methods. Sonication is based on the shear forces created by high frequency ultrasonic
vibrations generated by resonation (15–25 kHz) of a tuned probe or horn. The sonic pressure waves
generate shock waves with sufficient energy to disrupt cell walls, and reduce viscosity by shearing nucleic
acids. High-pressure homogenizers and pressure extruders operate by forcing a pressurized cell
suspension through a narrow orifice valve. A Dounce homogenizer consists of a round glass pestle that is
manually driven into a glass tube in which cell or tissue suspensions are sheared by forcing them through
a narrow space. It is a common practice to include multiple cell disruption methods to increase the
selectivity of product release, to increase the rate and yield of extraction, to minimize product damage,
and to reduce viscosity for downstream processes.

Procedure:
1. Inoculate 50 mL autoclaved LB media with the bacterial cells and incubate overnight in an orbital
shaker at 120 RPM.
2. Harvest the cells by centrifugation at 9000 rpm for 5 minutes in a centrifuge at 4oC
3. Remove and discard the spent medium by aspiration.
4. Resuspend the cell pellets in 2 mL of Phosphate buffered saline (PBS) and harvest the cells by
centrifugation as given in step 2
5. For the objective (A) resuspend the cell pellets in 500 µL of ice cold lysis buffer
6. Mix the sample (A) by pipetting and incubate on ice for 1 hr
7. For the objective (B&C) resuspend the cell pellets in 500 µL of ice cold lysis buffer containing 0.1%
Triton X-100 and 1 mg/mL Lysosyme taken separately in two different centrifuge tubes.
8. Mix the sample (B) by pipetting and disrupt the cells by ultrasonication with ten pulses of 30 s each at
30 s intervals.
9. Mix the sample (C) by pipetting and homogenize the cells in a dounce homogenizer for 20 minutes on
ice and the subject the cells to ultrasonication with ten pulses of 30 s each at 30 s intervals.
10. Clear all the lysates by centrifugation at 9000 rpm for 10 min at 4 °C.
11. Separate the supernatant from the cell debris
12. Remove a 2-10 µL sample of the supernatant for analysis of soluble proteins by Bradford assay.
13. Estimate the total soluble protein released after cell disruption.

Discussion:
Expt: 5 Over expression of a recombination protein in E. coli (BL21)

Aim: Aim is to identify the parameters for the optimal expression of the IPTG inducible recombinant
protein in E. coli

Introduction:

The vast majority of protein purification is now done with cloned, recombinant proteins expressed in a
suitable host. The predominant host is Escherichia coli. Many systems will allow expression levels of the
target protein of 10–40% of the total cell protein. Usually one carries out a test to see at what temperature
to grow the cells, how much inducer to use, and how long after induction you should harvest the cells.
The recombinant protein of our interest is cloned in pET15b vector. The pET vectors are a family of
expression vectors that utilize phage T7 promoters to regulate synthesis of cloned gene products that are
inducible by IPTG.

Principle: The lac promoter is the sequence that controls transcription of the lacZ gene coding for β-
galactosidase. Any general-purpose vector designed for blue/white screening for clones containing inserts
of foreign DNA can be used to express a foreign protein, usually as a fusion protein with amino acids
encoded by the amino terminus of the lacZ gene and/or the polylinker sequence. The pET series of
vectors originally developed by Studier et al. and since expanded, allow regulated expression of foreign
genes by bacteriophage T7 RNA polymerase. These vectors typically carry the colicin El (colEl) replicon
of pBR322 and confer resistance to ampicillin or kanamycin which can be used as a selection marker.
Their multiple cloning sites allow an inserted coding sequence to be placed under control of the "natural"
promoter for T7 RNA polymerase, or under the control of the so-called "T7lac" promoter, a derivative of
the natural promoter that has the lac operator (lacO) placed so that binding of the lac repressor blocks
transcription initiation. The lac promoter is induced by isopropylthiogalactoside (IPTG), so addition of
this chemical into the growth medium switches on transcription of a gene inserted downstream of the lac
promoter carried by the expression vector.

Procedure:

1. Inoculate 5-ml cultures (LB containing 50 μg/mL ampicillin) with 1 or two colonies of the E. coli
cells containing the expression plasmid. Incubate the cultures overnight at 37 oC with shaking.
2. Inoculate 3 test tubes with 7 ml of LB Medium containing 50 µg/mL ampicillin with 100 µL of
the overnight culture. Incubate the culture for greater than 2 hrs at 37 oC in a shaking incubator
until cell reach mid-log growth phase (A550 of 0.5-1.0).
 3. Transfer 1 ml of the uninduced culture (Zero time aliquot to microfuge tubes), measure the A 550
in a spectrophotometer, and centrifuge the tubes at maximum speed for 1 min at room
temperature in a microfuge. Remove the supernatant by aspiration.
4. Resuspend the pellet in 50 µl of 1x SDS gel-loading buffer, and heat the samples to 100 oC in
water bath for 3 minutes. Centrifuge the tubes at maximum speed for 2 minute at room
temperature in a microfuge, and store them on ice until all of the samples are collected and ready
to load on.
5. Induce the remainder of each culture by adding IPTG to a final concentration of 0.5 and 1.0 mM
and continue incubation at 37oC with aeration and shaking.
(Note: the concentration of IPTG used to induce lac repressor-regulated promoters can
dramatically influence expression. The highest concentration of IPTG that is used is 1mM and
optimum concentration varies between 0.01 and 5 mM. With some proteins, it is important to
induce transcription of the expression plasmid slowly with low IPTG concentration so as not to
overload the biosynthetic machinery of the bacterium).

6. At various time points during the induction period (e.g., 2, 4, and 6 hours), transfer 1 ml of each
culture to a microfuge tube, measure the A550 in a spectrophotometer, and centrifuge the tubes at
maximum speed for 1 minute at room temperature in a microfuge. Remove the supernatants by
aspiration.
7. Immediately process the pellet as described in the step 4.
8. After collecting all the samples, warm them to room temperature and load 0.15 OD550, units (of
original culture) or 40 µg of each suspension on a 10% SDS-polyacrylamide gel.
9. Run the gel at 8-10 V/cm until the bromophenol blue reaches the bottom of the stacking gel and
then increase the current to 12-18 V/cm until the bromophenol blue reaches bottom of the
resolving gel.
10. Stain the gel with Coomassie Brilliant Blue to visualize the induced protein.

Discussion and conclusions:

Over expression and Purification of the recombinant Proteins from Inclusion Bodies

Inclusion-body solubilization buffer I

50 mM Tris-Cl (pH 8.0)
1 mM EDTA (pH 8.0)
100 mM NaCl
8 M urea
0.1 mM PMSF
Prepare the buffer fresh just before use.
Expt: No. PROTEIN PURIFICATION BY SALT PRECIPITATION

Aim:
A) To separate a mixture of the given proteins
B) to characterize these proteins based on their solubilities as a function of salt concentration.

Introduction:
Structurally, proteins are the most complex biomacromolecules. This structural complexity is
due to the composition and sequence of the amino acids that make up proteins. The composition
and sequence of amino acids is different for every protein. As such, the unique chemical and
physical characteristics of a protein can be used to isolate it from other cellular components
using basic chemical techniques. Several different methods are available to separate proteins
from other proteins or biomolecules. The two most widely used are the precipitation methods
like (1) ammonium sulfate precipitation and (2) polyethyleneimine (PEI) precipitation. Over the
last hundred years, ammonium sulfate (AS) has remained the most widely used method for
protein precipitation. Polyethyleneimine (PEI) technique is especially popular for acidic proteins.
In this experiment the target proteins will be precipitated using the ammonium sulfate
precipitation technique. Ammonium sulfate fractionation is often used as the first purification step
in protein isolation. Different proteins are soluble to varying degrees in ammonium sulfate; some
proteins may precipitate at 30% saturation while others may remain soluble at 80% saturation.

Principle:
While several salts can be used as precipitants, AS has several properties that make it the most
useful. It is very stabilizing to protein structure, very soluble, relatively inexpensive, pure
material is readily available, and the density of a saturated solution (4.1 M) at 25 oC ( = 1.235
g/cm3) is comparatively lesser than other salting-out agents.

Ammonium sulfate solubility curve for a hypothetical protein. This represents the log solubility of a
hypothetical protein as a function of percent saturation of ammonium sulfate.
1. The figure shows a typical protein solubility curve where the log of the protein solubility is plotted as
a function of AS concentration.
2. The main features of this curve are a region at low salt where the solubility increases (called ‘‘salting
in’’), and then a region where the log solubility decreases linearly with increasing AS concentration
(called ‘‘salting out’’).
3. The latter part of the curve can be described by the equation
log10S =  -Ks( /2)
where, S in the solubility of the protein in mg/ml of solvent, /2 is the ionic strength, and  and Ks are
constants characteristic of the protein in question.

4. Suppose that the curve in the given figure is valid for our protein and that the concentration of the
targeted protein in a cell extract is 1 mg/ml.
5. The upper horizontal dotted line intercepts the solubility line at log10S 0 (S=1 mg/ml) and at an AS
percent saturation of 26%. This means that if you add AS to 26% saturation, all of your protein would
be soluble.
6. Now if you increased the AS to 32% saturation (the middle horizontal dotted line), the log S would
be -1 (S=0.1 mg/ml) so 90% of your protein would become insoluble and precipitate out.
7. For this extract, an excellent strategy would be to make a 26–32% saturated AS cut: add AS to 26%,
spin out insoluble material, and then make the supernatant 32% saturated and collect what
precipitates, which would contain 90% of your protein.
8. You would remove those proteins and cell components that precipitate at 26% saturation and those
that fail to precipitate at 32% saturation.

Usually we never get a curve like that shown in the figure for our protein of interest so we have
to determine the appropriate AS concentrations experimentally as described in the Experimental
Procedure section.

Materials and reagents required

i. Tris buffer 50mM and pH 9.5
ii. Caesin solution (1%, 2%) to be taken in 3 test tubes
iii. BSA solution 1% to be taken in 3 test tubes
Procedure:
While there are numerous variations on AS precipitation, the most common ones are to add solid
AS to a protein extract to give a certain percent saturation. Adding an amount of solid AS based
on Table given below is convenient, reproducible, and practical.
o
Final concentration of ammonium sulfate: Percentage saturation at 0 C (LHS)

Ref: Methods in Enzymology Volume 463, Guide to Protein Purification, Edited by

Richard R. Burgess and Murray P. Deutscher

1. Generally one determines a lower percent saturation at which the protein of interest just does
not precipitate and a higher percent saturation that gives >90% precipitation as described in
the section below.
2. Add solid AS to reach the lower value. Take care to add the AS slowly with rapid stirring so
that the local concentration does not ‘‘overshoot’’ the target value. Once the AS is
completely dissolved, allow the precipitation to continue for about 30 min.
3. This is a compromise between waiting several hours as precipitation slowly approaches
equilibrium and the desire to move along with the purification and not to introduce long
delays in the procedure.
4. Generally, one carries out all operations in an ice bucket or cold room.
5. Centrifuge at about 10,000x g for about 10 min in a precooled rotor to pellet the material that
is insoluble.
6. Carefully pour off the supernatant and determine its volume.
7. Determine the grams of AS from the lower desired percent saturation to the final higher
percent saturation.
8. Again add the AS slowly with rapid mixing to avoid high local concentrations and let the
solution sit for 30 min to allow precipitation to occur.
9. Centrifuge as above. Let the pellet drain for about 1 min to remove as much as possible of
the supernatant.
10. If you have carried out the test precipitation carefully, the pellet will contain 90% or more of
your target protein. This protein can be dissolved in an appropriate buffer and after either
dialysis, desalting, or dilution used in the next step of the purification.
 The optimal AS precipitation conditions can be determined by the following procedure:

1. Place a volume of the protein solution equivalent to 5 ml in each of a 15 mL centrifuge tube

2. Now add with mixing amounts of solid AS to give 15%, 25%, 35%, saturation based on the
AS saturation Table.
3. Incubate it for 30 minutes on ice to allow precipitation, and then centrifuge to pellet the
insoluble material.
4. The pellets represent the 15%, 25%, and 35% saturated AS pellets.
5. The volumes of the corresponding supernatants are determined and again solid AS is added
to raise each to a 10% higher level of saturation.
6. Again mix the solution, allow 30 min to precipitate, and then spin.
7. The three pellets are the 15–25
25%, 25–35% and 35–45% AS cuts.
8. Each of these is dissolved in buffer and assayed for total protein and subjected to SDS gel
analysis.

Scheme to determine the optimal AS cut to precipitate our target protein

 Isolation and Purification of Lysozyme from Chicken egg white using Ion-
exchange chromatography
Aim :

Aim of the experiment is to isolate and purify Lysozyme from Chicken egg white
using cation exchange resin and to analyze the isolated protein using SDS-PAGE.

Introduction
Lysozyme, also known as muramidase or N-acetylmuramide glycanhydrolase is an antimicrobial
enzyme produced by animals that forms part of the innate immune system. It is a special enzyme
found in tears, saliva, sweat, and other body fluids and mucosal secretions. It was the famous scientist
Alexander Fleming who discovered lysozyme. While growing a bacterial culture, a drop of mucus
from his nose fell into the culture. Eventually, he noticed that the bacteria in this culture had been
killed. He named the substance lysozyme by combining two words: 'lyse' and 'enzyme'. It is a single
chain polypeptide of 129 amino acids cross-linked with four disulfide bridges. It is a glycoside
hydrolase that catalyzes the hydrolysis of β(1→4) linkages between N-acetylmuramic acid and N-
acetyl-D-glucosamine residues in peptidoglycan, which is the major component of gram-positive
bacterial cell wall. Lysozyme is used for bacterial cell lysis in laboratories for protein extraction, in
conjunction with EDTA to lyse Gram-negative bacteria in which EDTA chelates metal ions in outer
membrane for easier degradation of peptidoglycan layer. Lysozyme has a high pI at pH 11.35 and a
low molecular mass of 14307 Da. These factors enable the purification of lysozyme by ion-exchange
chromatography. In egg white, lysozyme constitutes only about 10% of the total protein. Also,
lysozyme when at pH near 10.0 will uniquely be at a positive charge. This can be demonstrated by
performing an isoelectric focusing of crude protein extract from egg white in which all other proteins
will stop by around pH 9.5. So, with these two facts we can use a strong cation exchanger (SCX) like
carboxymethyl (CM) cellulose to isolate the lysozyme from other egg proteins.

Principle behind the isolation of lysozyme:

Isoelectric Point (PI) of an amino acid or any substance is the pH at which the net charge is
zero and the substance will not migrate in any direction under the influence of electric charge. Below
the isoelectric point the protein will have a net positive charge and above the isoelectric point the
protein it will have a net negative charge. Since, the isoelectric point of Lysozyme is 11.35, at a pH
between 9.5 and 10.0 it will have a net positive charge. Ion -exchange chromatography is the method
of separation of molecules according to the charge. Ion exchange resins like Carboxymethyl
Sepharose (CM Sepharose) are cationic exchanger that traps positively charged molecules. Thus,
positively charged proteins are bound to the CM sepahrose and are released by altering the ionic
strength of the eluting buffer containing sodium chloride.

Methods
1. Carefully remove the egg white from two fresh Chicken eggs.
2. Prepare the egg white mixture by diluting with five volumes of 25 mM glycine, pH 9.2;

3. Shear the mixture and homogenize gently with a teflon-glass homogenizer,

4. Strain through four layers of cheesecloth, and finally, filter through a fast-flow filter paper.
 5. Load 5 mL of this filtered mixture onto a 3-mL bed of the cation exchange resin equilibrated
with the glycine buffer and wash it on with 4 mL of glycine buffer. (OR) 5mL of the filtered
mixture is stirred gently with 3 mL of the cation exchange resin for 3-4 hours in cold and then
loaded on to a glass column

6. Then elute the column with 4 mL portions of glycine buffer containing 0.10 M NaCl to
remove the non-specific binding

7. Then elute the column with 0.75 M NaCl and collect the eluate in small fractions in clean
glass tubes.

8. All the aliquots are pooled to get a purified solution of lysozyme and protein concentration is
estimated by performing a Bradford assay.
9. Purity of the extract is checked by performing SDS-PAGE by running equivalent volumes
from Fractions 1, 2 and 3 to compare the bands.

Reagents required:
Glycine-NaOH Buffer (25 mM Glycine, pH 9.2)

Stock solutions:

200 mM Glycine buffer pH 9.2

NaCl stock solution 2 M

Results:
Elution profile

S. No. Sample Number Volume Absorbance

(Bradford Reagent)

Bed Volume of resin:

Volume of washing buffer:

Volume and Concentration of the pooled fractions:

Yield:

Discussion:
Production of citric acid in batch culture fermentation by Aspergillus niger and
recovery of citric acid from the culture broth:

Aim: Aim of the experiment is to produce citric acid by batch culture fermentation using
Aspergillus niger and recover the citric acid and report the findings.

The above objective will be achieved by the following steps which are divided into 3 parts.
Step Objective Part
No.
1 Experimental design and planning
2 Preparation of reagents, sporulation media and inoculation A
on sporulation media
3 Preparation of production media, spore suspension and
inoculation B
4 Sample collection and biomass determination
5 Determination of Citric Acid production and carbohydrate
consumption C
6 Isolation of citric acid

Introduction: Citric acid occurs naturally as a component of many fruits. This acid is also
produced by chemical synthesis and fungal fermentation. Chemical synthesis has proved
uneconomical due to the fact the starting materials are worthier than the end product. Most of
the commercial citric acid in the world is produced by fungal fermentation due to its
economical competitiveness. Commercial citric acid has wide application. It is employed as
an acidulant in the food and pharmaceutical industries, and it finds extensive use in the
production of carbonated beverages. It is also employed commercially as a chelating and
sequestering agent, and citrate and citrate esters are used as plasticizers.

Principle: Citric acid accumulates during the controlled fermentative growth of particular
species of Penicillium and Aspergillus. Citric acid is an intermediate component of the TCA
cycle. However, the high-percentage conversions of carbohydrate observed in the formation
of these acids by fungal fermentation require that the acid accumulation proceed via routes
other than or in addition to the TCA cycle. Although the mechanism is not fully understood,
it has been reported that the organic acid accumulation is associated with an apparent
nutritional deficiency which is imposed by the fermentation conditions. Citric acid excretion
in large amounts by A. niger only occurs when certain aspects of cell growth is not optimal. It
has been shown that high levels of citric acid accumulated in the absence of manganese, zinc,
high initial sucrose concentration and aeration. Low pH is important for the accumulation.
The ability of A. niger to excrete citrate depends on many variables, both genetic and
environmental. Some strains are good citrate accumulators, while other strains do not
accumulate at all. Citrate accumulation property may be induced by mutation.

Part A:
Experimental design and planning
The following media and reagent have to be prepared before starting the experiment
1. Dinitrosalicyclic acid (DNA) reagent,
2. Potato dextrose Agar (PDA) sporulation media
 3. Production media
4. Autoclaving the sporulation media, production media and other test tubes, centrifuge
tubes and other glassware required for the experiment
5. Inoculation of A. niger spores from the stock culture on sporulation media and
allowing it to grow for 7 days
6. Preparation of spore suspension in autoclaved distilled water containing 0.5 % Tween
80.
7. Inoculating the spore suspension in 50 mL of production media
8. Samples will be collected from the 4th day onwards for the estimation of biomass,
citric Acid production and carbohydrate consumption
9. Isolation of citric acid
10. Decontaminating the test tubes and other glass ware by Autoclaving after completion
of the experiment

2. Preparation of sporulation media:

Procedure:

1. Sporulation media: Potato Dextrose Agar (PDA) media:

Potatoes 200g
Dextrose 20g
Agar 20g

Potato infusion was prepared by boiling peeled potatoes and straining the broth through
cheesecloth. To this dextrose and agar were added. The media was autoclaved, and slants
prepared.

A loopful of spores were inoculated onto the slants, and left undisturbed for 7 days.

Discussion:
Experiment:
Production of citric acid in batch culture fermentation by Aspergillus niger and
recovery of citric acid from the culture broth: Part B

Aim: Aim of the experiment is (1) Preparation of production media, spore suspension and
inoculation (2) Sample collection and biomass determination

(1) Preparation of production media, spore suspension and inoculation

Preparation of production media:

Composition of the production media

Sucrose 12%
NH4NO3 0.2%
KH2PO4 0.1%
MgSO4.7H2O 0.2%
pH 3.0-3.5 (add HCL for pH adjustment)

Procedure for the Preparation of spore suspension and inoculation in the production
media
1. Prepare a Aspergillus niger spore suspension by adding5 ml sterile 0.5% sterile
Tween 80 per slant. Tween 80 is a non-ionic detergent which serves as a wetting
agent to produce a homogeneous suspension of highly hydrophobic spores.
2. Take 20 ul of the spore suspension and count the spores using a Neubauers’s chamber
after appropriate dilution.
3. Take 50 mL of production media in a 250 mL conical flask and sterilize by
autoclaving.
4. Add 0.2 ml Tween 80 spore suspension to each flask (104 to 105 spores/ml, ~600 ul
for 50 ml culture). Be careful not to puncture the agar.
5. Incubate flasks (properly labelled) on culture rotator at 30 oC incubator at 150 rpm.
6. No samples are collected till the end of 4 days
7. Collect samples from the 5th day to 9th day to estimate biomass, citric acid production
and carbohydrate consumption.

(2) Sample collection and biomass determination

1. Take 10 mL of inoculated cultures (all liquid and fungal growth) into one sterile
centrifuge tube.
2. Centrifuge at 10000 rpm for 10 min.
3. Pour clear supernatant into a labeled culture tube (not sterile).
4. Put on ice.
5. Often the fungal pellet is loose. Stop collecting the supernatant if the pellet is going to
be transferred. Any extra liquid will be evaporated upon drying and contribute little to
cell mass.
6. Weight and record the biomass of the fungal pellet collected on each day after drying
the samples at 100oC for 22 h.
7. Discard the pellet after weighing.

Discussion:
Experiment:
Production of citric acid in batch culture fermentation using Aspergillus niger and
recovery of citric acid from the culture broth: Part C

Aim of the experiment is (1) Determination of Citric Acid production and carbohydrate
consumption (sucrose and glucose) and (2) Recovery of the citric acid from the culture broth

(1a) Determination of citric acid by titration method

Aim:
To determine citric acid production of Aspergillus niger by titration with NaOH.
Principle:
Titration is a common laboratory method of quantitative/chemical analysis that can be used
to determine the concentration of a known reactant (analyte). The basis of the method is a
chemical reaction of a standard solution (titrant) with a solution of an analyte. The analyte
(A) is a solution of the substance whose concentration is unknown and sought in the analysis.
The titrant (T) is a solution in which the concentration of a solute is precisely known.
Citric acid is a weak acid. Titration of weak acids with sodium hydroxide gives salts which
will be hydrolysed in aqueous solution to a greater or lesser extent depending upon the
dissociation constant of the acid. The pH of the solution at the equivalence point will be
above 7, and the indicator most frequently used in such titrations it phenolphthalein. At end
point the number of moles of titrant is equal to the number of moles of analyte or some
multiple thereof.

Chemical reaction:

Citric acid Sodium Hydroxide Sodium citrate water

192.12 g of citric acid  3000 ml of 1 N NaOH

32.02 g of citric acid  1000 ml of 0.5 N NaOH
 Each ml of 0.5 N NaOH  0.032 g of Citric acid

Materials required:
Burette , conical flasks, glass pipette, reagent bottles (stock solutions), beaker, distilled water,
phenolphthalein,
analyte: citric acid
titrant: sodium hydroxide (0.5 M)
Procedure:

1. Fill the titration flask with VA=5 ml of the sample.

2. Add to the solution of acid 1-2 drops of phenolphthalein solution.
3. Fill the burette with a standard solution of 0.5 N NaOH (titrant).
4. Perform the titration.
5. When the endpoint of titration has been reached, read the used volume of NaOH from
the burette (VT). Write it down to the table.
6. Repeat the procedure 2 times.
7. Determine the average volume of titrant from four titrations V T (ml).
8. Using the above mathematical equation calculate the percentage concentration of
Citric acid in the sample.
Observation table:
Sample Titrant Volume of titrant VT CA (M)
Volume VA concentration (ml) VT (ml)
(ml) CT (M)

(1b) Determination of glucose by dinitrosalicylic acid method (DNS)

Reagents:

(A) Dinitrosalicylic Acid Reagent Solution, 1% (DNS)

Composition of the reagent:

o Dinitrosalicylic acid: 10 g
o Phenol: 2 g (optional, Phenol, up to 2g/l, intensifies the color density. It
changes the slope of the calibration curve of absorbance versus glucose
concentration but does not affect the linearity.)
o Sodium sulfite: 0.5 g
o Sodium hydroxide: 10 g
o Add water to: 1 liter

(B) Potassium sodium tartrate solution, 40%

Procedure:

1. Add 3 ml of DNS reagent to 3 ml of glucose sample in a lightly capped test tube. (To
avoid the loss of liquid due to evaporation, cover the test tube with a piece of paraffin
film if a plain test tube is used.)
2. Heat the mixture at 90º C for 5-15 minutes to develop the red-brown color.
3. Add 1 ml of a 40% potassium sodium tartarate (Rochelle salt) solution to stabilize the
color.
4. After cooling to room temperature in a cold water bath, record the absorbance with a
spectrophotometer at 575 nm.

(1c) Determination of sucrose by dinitrosalicylic acid method (DNS)

The DNS method that is used for the estimation of glucose can be modified to estimate
sucrose concentration. Sucrose is first hydrolyzed in an acid solution to yield glucose and
fructose, then the solution is neutralized with KOH then the original DNS method for glucose
estimation was followed.

Procedures

1. Add 1 drop, or 20 µl, of concentrate HCl (37.3%, 11.9 N) solution to 1 ml of the

sucrose solution. Allow the hydrolysis to proceed at 90ºC for 5 minutes.
2. Add 3 drops, or 0.05 ml, of the 5 N KOH solution to neutralize the acid, because the
DNS method must be applied in an alkaline condition to develop the red brown color
which represents the presence of reducing sugars.
3. Add the DNS reagent and follow the DNS method henceforth.
4. Generate a calibration curve to correlate the absorbance to the sucrose concentration.

The difference in the absorbance between the acid treated sample and the untreated sample is
due to the presence of sucrose. The sucrose concentration can then be calculated from a
calibration curve based on that difference in the absorbance.

(2) Recovery and purification of citric acid

1. Harvested broth

2. Filter off A. niger mycelium using a rotary vacuum filter.

3. Add Ca(OH)2 to the filtrate until pH 5.8

4. Calcium citrate

5. Add sulfuric acid while at 60oC

6. Filter on rotary vacuum filter to CaSO4 and wash it with water several times.


 7. Treat it with activated charcoal to decolorize

8. Pass it through cation and anion exchange resins to demineralize it

9. Evaporate to point of crystallization at 36 oC

10. Crystals of citric acid monohydrate will separate out.

11. Dry the crystals at 50-60 oC

Discussion: