DePaul Discoveries DePaul Discoveries

Volume 3 Issue 1 Article 10

2014

Generation of Chimeric Antibody Light Chain Plasmid Generation of Chimeric Antibody Light Chain Plasmid

Lily Arendt Larendt1@mail.depaul.edu

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Recommended Citation Recommended Citation Arendt, Lily (2014) "Generation of Chimeric Antibody Light Chain Plasmid," DePaul Discoveries: Vol. 3 : Iss. 1 , Article 10. Available at: https://via.library.depaul.edu/depaul-disc/vol3/iss1/10

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Generation of Chimeric Antibody Light Chain Plasmid

Lily Arendt*

Department of Chemistry, DePaul University

ABSTRACT In order to determine the distance between the antigen-binding site and the crystallizable

fragment (Fc) region of an antibody using single-molecule Förster Resonance Energy Transfer, dyes must

be attached to these locations. To accomplish this, the variable regions of the light and heavy chains of an

antibody can be modified to introduce dye-reactive amino acids. Initial attempts to alter the variable light

chain (VL) gene included trying to ligate a 350 bp variable gene into an 11,000 bp plasmid. When these

attempts were unsuccessful, the variable light chain gene was ligated into a commercially available 3500

bp plasmid.

INTRODUCTION

Antibodies are comprised of four polypeptide

chains including two identical light chains and

two identical heavy chains (Figure1).1 One end

of each of the two heavy chains interacts with

the other to form the crystallizable fragment (Fc)

region of the antibody, which is the lower lobe

and the other ends interact with the light chains

to form the upper lobes. The two upper lobes are

separated from the lower lobe by an unstructured

hinge region. The presence of this hinge region

provides segmental flexibility between the lobes

____________________________________

*Faculty Advisor: Dr. Cathrine A. Southern

Department of Chemistry

Research Completed in Autumn 2013

Author Contact: larendt1@mail.depaul.edu

and grants antibodies a larger range to search for

antigens.2

Binding of effector molecules to the

Fc region can result in an immune response

within the body. The two upper lobes of the

antibody are where the fragment antigen-binding

(Fab) regions reside. If the two Fab regions

engage a single antigen, the possibility of

forming an immune complex, or a group of

antibodies bound to the antigen, is increased.

The formation of immune complexes is

necessary for the generation of an immune

response and it is affected by antibody

flexibility, or the ability of an antibody to take

on a variety of structures. Further researching

the possible antibody conformations will lead to

a better understanding of the variety of

structures antibodies exhibit.

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Arendt: Generation of Chimeric Antibody Light Chain Plasmid

Published by Via Sapientiae, 2014

METHODS

Examining the distance between the Fc and Fab

regions can reveal details of antibody structures.

This distance can be seen in crystal structures,

which provide information about one particular

antibody conformation.1 It is of particular

interest to determine whether or not on

structure is preferred or if a multitude of Fab

distances are possible. Discovering how this

distribution of conformations is affected when

Fc receptor binding occurs is also of interest.

This information on the structure of the IgG

antibody could lead to an enhanced

understanding of the medicinal properties of

antibodies.

The technique of single-molecule Förster

Resonance Energy Transfer (FRET) can be used

to determine the distance between the Fc and

Fab regions. In order to use the technique, dyes

Figure 1: Crystal structure of an IgG antibody. The

Fc region is attached to the upper lobes by a hinge

region. Antibody drawn from the PDB file 1IGT.

Harris, L.J.; Larson, S.B.; Hasel, K.W.; McPherson,

A. (1997) Biochemistry 36, 1581.

Isolate Plasmid Open Plasmid via

Enzymes

ce between the Fc and Fab

regions can reveal details of antibody structures.

This distance can be seen in crystal structures,

which provide information about one particular

It is of particular

interest to determine whether or not one

structure is preferred or if a multitude of Fab-Fc

distances are possible. Discovering how this

distribution of conformations is affected when

Fc receptor binding occurs is also of interest.

This information on the structure of the IgG

d to an enhanced

understanding of the medicinal properties of

molecule Förster

Resonance Energy Transfer (FRET) can be used

to determine the distance between the Fc and

Fab regions. In order to use the technique, dyes

molecules must be attached to those regions.

The dyes cannot be attached unless both the

variable light (VL) and var

chains are modified and dye

acids are introduced in the appropriate locations.

Through past experiments the variable heavy

chain and the Fc region have been mutated and a

dye-reactive cysteine residue was introduced in

the Fc region. The goal of this experiment was

to add a dye-reactive lysine residue to the Fab

region by manipulating the variable light chain

gene. In order to introduce the

VL gene from a mouse antibody was inserted

into a plasmid containing the other genes for the

light chain through ligation. The process of

ligation involved isolating the original plasmid,

opening it through the use of enzymes, and

adding in the new VL region (Figure 2).

new region was taken from a mouse antibody

because it would produce an antigen

containing a reactive lysine residue, which is

necessary for dye attachment.3

Once dye molecules are attached to any location

on the antibody, its structure can be examined

using FRET. This technique is based on a weak

dipole-dipole coupling involving two different

dye molecules, a donor and an acceptor, which

are attached at two different points on an

antibody. The process begins by the excitatio

of the donor molecule, or having the donor

molecule absorb light, by means of a laser. If

there is no acceptor molecule present, no

transfer of energy occurs and the donor molecule

will fluoresce, or give out some of the absorbed

energy in the form of light. If energy is

transferred to an acceptor molecule, the acceptor

will fluoresce. The relative intensity of the

fluorescence signals from the donor and

acceptor depends on the distance between the

two dye molecules, meaning that the closer they

are to one another, the higher the energy transfer

Crystal structure of an IgG antibody. The

Fc region is attached to the upper lobes by a hinge

region. Antibody drawn from the PDB file 1IGT.

Harris, L.J.; Larson, S.B.; Hasel, K.W.; McPherson,

Open Plasmid via

Enzymes

Add New VL Region

Figure 2:

representation of the

insertion of a new V

into a plasmid.

molecules must be attached to those regions.

The dyes cannot be attached unless both the

) and variable heavy (VH)

chains are modified and dye-reactive amino

acids are introduced in the appropriate locations.

Through past experiments the variable heavy

chain and the Fc region have been mutated and a

reactive cysteine residue was introduced in

c region. The goal of this experiment was

reactive lysine residue to the Fab

region by manipulating the variable light chain

gene. In order to introduce the lysine residue, a

gene from a mouse antibody was inserted

the other genes for the

light chain through ligation. The process of

ligation involved isolating the original plasmid,

opening it through the use of enzymes, and

region (Figure 2). This

new region was taken from a mouse antibody

se it would produce an antigen-binding site

containing a reactive lysine residue, which is 3

Once dye molecules are attached to any location

ts structure can be examined

using FRET. This technique is based on a weak

dipole coupling involving two different

dye molecules, a donor and an acceptor, which

are attached at two different points on an

antibody. The process begins by the excitation

of the donor molecule, or having the donor

molecule absorb light, by means of a laser. If

there is no acceptor molecule present, no

transfer of energy occurs and the donor molecule

will fluoresce, or give out some of the absorbed

ght. If energy is

transferred to an acceptor molecule, the acceptor

will fluoresce. The relative intensity of the

fluorescence signals from the donor and

acceptor depends on the distance between the

two dye molecules, meaning that the closer they

e another, the higher the energy transfer

Figure 2: A schematic

representation of the

insertion of a new VL region

into a plasmid.

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DePaul Discoveries, Vol. 3 [2014], Iss. 1, Art. 10

https://via.library.depaul.edu/depaul-disc/vol3/iss1/10

efficiency. Single molecule FRET is beneficial

because it allows the distances between the dye

molecules on many individual molecules to be

examined. A histogram can then be constructed

with a distance value for each molecule instead

of simply the average distance i

experiment.

RESULTS

Initial attempts in the experiment included

amplifying the VL gene from a plasmid

containing a single chain variable fragment of a

mouse antibody called 84G3.3 The V

amplified using the polymerase chain reaction

(PCR). A ligation reaction between the V

and an 11,000 bp plasmid was carried out. To

determine if the ligation was successful,

restriction enzymes were used to cut the ligation

product to see if the expected 350 bp V

be observed. When analyzed on an agarose gel,

the results indicated that the ligation was

unsuccessful. The lack of success was attributed

to the difficulty involved in ligating a very small

fragment into a much larger one. To avoid this, a

commercially available pFuse 3500 bp plasmid

was then purchased, and was opened at the

EcoRI and BsiWI restriction enzyme cut sites in

order to allow for ligation of the VL

3).

Figure 3: Plasmid map for the pFuse light chain. The

pFuse map shows the locations of the EcoRI and

restriction enzyme cut sites where the VL gene was

inserted.

. Single molecule FRET is beneficial

because it allows the distances between the dye

molecules on many individual molecules to be

examined. A histogram can then be constructed

with a distance value for each molecule instead

of simply the average distance in the

nitial attempts in the experiment included

gene from a plasmid

containing a single chain variable fragment of a

The VL gene was

amplified using the polymerase chain reaction

(PCR). A ligation reaction between the VL gene

and an 11,000 bp plasmid was carried out. To

ermine if the ligation was successful,

restriction enzymes were used to cut the ligation

product to see if the expected 350 bp VL would

be observed. When analyzed on an agarose gel,

the results indicated that the ligation was

ess was attributed

to the difficulty involved in ligating a very small

fragment into a much larger one. To avoid this, a

commercially available pFuse 3500 bp plasmid

was then purchased, and was opened at the

WI restriction enzyme cut sites in

L gene (Figure

The newly ligated plasmid was then transformed

into E. coli cells and the DNA was replicated.

The DNA from colonies that grew was then

isolated. PCR was performed on the new ligated

pFuse plasmid, and the results were run on an

agarose gel. A band at the expected 350 bp mark

for the VL gene was observed, showing that the

ligation was successful (Figure 4).

CONCLUSION

After many attempts at ligating a V

into an 11,000 bp plasmid, the V

of the single chain variable region of a

mouse antibody was successfully ligated

into the smaller 3500 bp pFuse plasmid.

The 350 bp band that appeared on the

agarose gel demonstrated this success and

the DNA can now be sent out for

sequencing. Following this, the antibody

DNA will be shipped to a company that

will express and purify the mutated

antibody. Dyes will then be attached to

the antibody, and single

experiments will be performed on the dye

labeled antibodies to determine the F

distances present.

: Plasmid map for the pFuse light chain. The

RI and BsiWI

gene was

350 bp ����

1 2 3 4 5 6 7 8 9

Figure 4: Stained agarose gel of the variable

light chain gene from the uncut ligated pFuse

plasmid. Lanes 1-4 and 6-9 are ligated PCR

products with amplified VL

Lane 5 is the ladder for base pair reference.

The newly ligated plasmid was then transformed

cells and the DNA was replicated.

The DNA from colonies that grew was then

isolated. PCR was performed on the new ligated

the results were run on an

agarose gel. A band at the expected 350 bp mark

gene was observed, showing that the

ligation was successful (Figure 4).

After many attempts at ligating a VL gene

into an 11,000 bp plasmid, the VL region

of the single chain variable region of a

mouse antibody was successfully ligated

into the smaller 3500 bp pFuse plasmid.

The 350 bp band that appeared on the

agarose gel demonstrated this success and

the DNA can now be sent out for

owing this, the antibody

DNA will be shipped to a company that

will express and purify the mutated

antibody. Dyes will then be attached to

the antibody, and single-molecule FRET

experiments will be performed on the dye-

labeled antibodies to determine the Fc-Fab

1 2 3 4 5 6 7 8 9

agarose gel of the variable

light chain gene from the uncut ligated pFuse

9 are ligated PCR

L region at 350 bp.

Lane 5 is the ladder for base pair reference.

3

Arendt: Generation of Chimeric Antibody Light Chain Plasmid

Published by Via Sapientiae, 2014

REFERENCES

Harris, L.J.; Larson, S.B.; Hasel, K.W.;

McPherson, A. (1997) Biochemistry 36,

1581.

Huang, G. Steven, Yu-Shiun Chen, and Hsiao

Wei Yeh. "Measuring the Flexibility of

Immunoglobulin by Gold

Nanoparticles." Nano Letters 6.11

(2006): 2467-471. Print.

Wagner, J.; Lerner, R. A.; Barbas, C. F.

Science (1995) 270, 1797-1800.

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DePaul Discoveries, Vol. 3 [2014], Iss. 1, Art. 10

https://via.library.depaul.edu/depaul-disc/vol3/iss1/10