Temperature-Triggered Purificationof Antibodies

Jae-Young Kim,1,2 Ashok Mulchandani,1 Wilfred Chen1

1Department of Chemical and Environmental Engineering,University of California, Riverside, California 92521;fax: 909-787-5696; e-mail: adani@engr.ucr.edu or wilfred@engr.ucr.edu2Environmental Toxicology Graduate Program, University of California,Riverside, California 92521

Received 21 July 2004; accepted 14 December 2004

Published online 30 March 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20451

Abstract: In this article the unique capability of elastin-likeprotein (ELP) to reversibly precipitate was combined withthe high affinity and specificity of antibody-binding do-mains such as Protein G, Protein L, or Protein LG as ageneral method for antibody purification that combinesin a unique manner the simplicity and robustness oftemperature-triggered precipitation with the selectivity ofaffinity interactions. In a single precipitation step, anti-bodies derived from different sources (animal sera or hy-bridoma cell cultures) were selectively recovered by asimple temperature trigger. Due to the versatility of thebinding ligands toward different classes of antibodies, webelieve that this technology will be useful as an econom-ical, highly efficient, and universal platform for thepurification of antibodies. B 2005 Wiley Periodicals, Inc.

Keywords: protein A; protein purification; affinityprecipitation

INTRODUCTION

Antibodies or immunoglobins (Ig), because of their highly

specific nature, are valuable tools for environmental moni-

toring and for in vitro and in vivo medical diagnostics

(Templin et al., 2003). Therapies based on antibodies have

also been gaining momentum for the prevention and treat-

ment of infectious diseases (Keller and Stiehm, 2000), for

protection against biological warfare agents (Maynard et al.,

2002), and as therapeutic agents for the treatment of dis-

eases like cancer (Carter, 2001).

Large-scale production of antibodies up to 1–2 g/L has

been accomplished using transgenic animals or by hybrid-

oma technology (Vandekerckhove et al., 1993; Mckinney

et al., 1995; Bibila and Robinson, 1995). Purification of

antibodies, however, presents an additional challenge due

to the broad range of sources such as blood, milk, cell

culture supernatant, low antibody concentration, excessive

amounts of contaminating proteins, and the requirement of

high purity. A method that will enable efficient and simple

recovery of antibodies will greatly minimize the overall

manufacturing costs.

Affinity chromatography based on immobilized anti-

body-binding proteins (either protein A, G, or L) is a

commonly used method for antibody purification (Fassina

et al., 2001; Huse et al., 2002). However, conventional

affinity chromatography is relatively expensive and re-

quires chemical coupling of the binding proteins onto a

solid support, which can cause a significant decrease in

their binding affinity toward antibodies. Protein A, for ex-

ample, when immobilized onto a rigid ceramic compo-

site lost 75% of its original IgG-biding capacity (Guerrier

et al., 1998).

Affinity precipitation is a solution-phase analog to

affinity chromatography, in which a thermally reversible

polymer, poly-N-isopropylacrylamide (PNIPAM), was

chemically conjugated to the binding proteins (Ding et al.,

1999; Kumar et al., 2001; Fong et al., 2002). Purification is

based on a simple environmental trigger in combination

with the specificity and affinity of the binding proteins.

Since the binding step is executed in the aqueous phase, no

mass transfer resistance or steric hindrance problems will

occur as in the case of affinity chromatography. Although

operationally simple (Chen and Hoffman, 1990), this meth-

od still remains tedious, as it requires complicated organic

synthesis as well as chemical coupling that decreases the

affinity of the binding proteins.

One way to circumvent these problems is to utilize ther-

mally responsive biopolymers capable of reversible phase

separation. Elastin-like polypeptide (ELP), consisting of

the repeating pentapeptide VPGVG, can undergo a revers-

ible phase transition from water-soluble forms into aggre-

gates similar to PNIPAM polymer within a wide range of

conditions that are controlled by the chain length and com-

position (Urry, 1992, 1997). The feature to reversibly ag-

gregate ELP above the transition temperature has been

demonstrated for protein purification (Shimazu et al., 2003;

Kostal et al., 2001, 2004; Meyer and Chilkoti, 1999; Meyer

et al., 2001; Trabbic-Carlson et al., 2004). In these cases,

fusion proteins with ELP were generated while retaining

B 2005 Wiley Periodicals, Inc.

Correspondence to: Ashok Mulchandani or Wilfred Chen

Contract grant sponsor: National Science Foundation

Contract grant number: EIA-0330451

their temperature responsiveness, as well as the function-

ality of the fusion partner.

Although ELP fusions could be similarly generated with

each individual antibody of interest, not all the genes

coding for different antibodies are currently available for

fusion construction. Even the use of a single-chain Fv

fragment rather than the full antibody may not be desir-

able due to the lower affinity. It is clear that a universal

method is needed to provide a cost-effective and efficient

means for the purification of any antibody of interest. In

this study, ELP fusions containing either Protein G or

Protein L were constructed, enabling rapid binding to any

Ig of interest and the capability to undergo a revers-

ible phase transition. The utility of these ELP fusions for

rapid, efficient purification of a wide range of antibodies

was demonstrated.

MATERIALS AND METHODS

Materials

The supernatant of hybridoma cell culture (C1B7) was

purchased from Developmental Studies Hybridoma Bank

(Iowa City, IA). IgGs, mouse and rabbit sera were pur-

chased from Sigma-Aldrich (St. Louis, MO). Goat anti-

mouse IgG-horseradish peroxidase (HRP) conjugate and

human IgM-HRP conjugate were purchased from Pierce

Biotechnology (Rockford, IL). Goat anti-mouse IgG-alka-

line phosphatase (AP) conjugate, AP reagent, and chloro-

naphthol were purchased from Bio-Rad (Hercules, CA).

Molecular Biology, Bacterial Strains, and Plasmids

DNA manipulations were performed according to standard

procedures unless specified otherwise (Sambrook and

Russell, 2001). PCR was performed using the Taq DNA

polymerase (Promega, Madison, WI) according to the

manufacturer’s instruction. E. coli JM109 (recA1 supE44

endA1 hsdR17 gyrA96 relA1 thi �(lac-proAB) FV [traD36

proAB+ lacIq lacZ �M15]) and BL21(DE3) (hsdS gal

(EcIts857 ind1 Sam7 nin5 lacUV5-T7 gene 1)) were grown

on LB agar for solid culture and in terrific broth for liq-

uid culture. All media contained 0.1 mg/mL of ampicillin

for selection. Plasmid pET-Ela78h6 (Kostal et al., 2001)

and plasmid pLG (Kihlberg et al., 1992) were used as the

sources of the ELP gene and the Protein G and L

gene, respectively.

Construction of Expression Vectors

The genes coding for Protein G and Protein L were am-

plified as 407-bp and 905-bp PCR fragments using prim-

er sets Upper-G (5V-tcc ccc ggg agg agg agg agg aac tta

caa att-3V):Lower-G (5V-tat ggt gac ctt cag gta ccg taa agg

tc-3V) and Upper-L (5V-tcc ccc ggg agg agg agg agg aaa aga

aga aac-3V):Lower-L (5V-tat ggt gac ctg caa atc taa tat taa

tag-3V). The Protein LG fragment was amplified as a 1319-

bp PCR fragment using the Upper-L and Lower-G primer

set. The PCR products were digested with XmaI and BstEII

and inserted into a similarly digested pET-Ela78h6, re-

sulting in pELP-ProG, pELP-ProL, and pELP-ProLG.

Expression and Purification of Fusion Proteins

Escherichia coli strain BL21(DE3) containing each plas-

mid was inoculated from a single colony and grown at

37jC and 300 rpm in 25 mL of terrific broth. After

48 h the culture was harvested and resuspended in 5 mL

of phosphate-buffered saline (PBS; 137 mM NaCl,

2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7).

Cells were disrupted for 5 min by a sonicator (Virtis, NY)

and the cell debris was removed by centrifugation for

15 min at 15,000g.

The inverse temperature transition was used for the pu-

rification of ELP fusions. To lower the transition temper-

ature and to facilitate precipitation, NaCl was added to a

final concentration of 1 M to the crude extract. The sam-

ples were heated to 37jC for 10 min and centrifuged at

15,000g at 37jC for 15 min. The pellets containing ELP

fusions were dissolved in ice-cold PBS and centrifuged

at 15,000g at 4jC for 15 min to remove any insoluble

proteins. This temperature transition cycle was repeated

once more and the pellets containing ELP fusions were

finally redissolved in ice-cold PBS. The purity of the pro-

tein was determined using 10% SDS-PAGE followed by

silver staining (Bio-Rad). Western blot was performed

using goat IgG-AP with the AP color reagent (Bio-Rad) for

G fusions and human IgM-HRP with HRP color reagent

(filtered 10 mL of 50 mM pH 7.6 Tris buffer with 3 mg of

chloronaphthol in 0.1 mL ethanol, containing 10 AL of 30%

H2O2) for L fusions.

Characterization of Fusion Proteins

The inverse transition temperature profiles of the ELP

fusions were determined spectrophotometrically in a 96-

well microplate reader (POLARstar Optima, BMG Lab-

technologies, Durham, NC). Turbidity measurements were

conducted at 620 nm from 25–40jC with 100 Al of 0.1 mM

of ELP or ELP fusions in PBS containing 0.5 M NaCl.

To demonstrate the binding of antibodies via the G or L

domains, 0.1 mg of each fusion protein in 0.1 mL of PBS

was immobilized onto a microtiter plate for 30 min at 37jC

based on the hydrophobic interaction between the fusion

and the polystyrene surface. After discharging solutions,

1:5,000 dilutions of 1 mg/mL of goat IgG-HRP, human

IgM-HRP, and HRP in PBS were added and incubated for

30 min at 37jC. The plate was washed three times with

37jC PBST (0.5% Tween-20 in PBS) and the amount of

bound IgGs was quantified by the conjugated HRP activity

by incubating with 0.1 mL of HRP substrate (20 mg of

o-phenylenediamine in 10 mL of 0.1 M pH 4.6 citrate-

phosphate buffer, containing 4 AL of 30% H2O2) for 10 min.

374 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 90, NO. 3, MAY 5, 2005

The absorbance was measured at 490 nm using a micro-

plate reader (Model 3550-UV, Bio-Rad).

IgG Purification

To demonstrate the purification of IgGs by temperature

precipitation, 1 mg of ELP-ProG was mixed with 0.5 mg

of purified mouse- or rabbit-IgG in 0.5 mL of PBS. The

mixture was incubated for 30 min at room temperature to

allow binding between Protein G and the IgGs. To recover

the ELP-ProG-IgG complex, 0.1 mL of 5 M NaCl was

added to the sample and incubated at 37jC for 5 min. After

centrifugation at 15,000g while maintaining the same tem-

perature, the supernatant (nonbound fraction) was dis-

charged and the pellet containing the ELP-ProG-IgG

complex was resolubilized with 0.1 mL of ice-cold PBS.

For elution of the IgG, 0.4 mL of 0.1 M sodium citrate

(pH 2.6) was added and the sample was stored on ice for

10 min. The eluted IgG was recovered by removing ELP-

ProG by inverse temperature transition at 37jC. Samples

collected at different stages were analyzed by nonreducing

10% SDS-PAGE, followed by silver staining.

For IgG purification from hybridoma cell culture, 1 mg

of ELP-ProG in PBS was mixed with the 450 AL of su-

pernatant (C1B7, 19.35 Ag of IgG) to a final volume of

0.5 mL. Again, recovery of ELP-ProG-IgG complex and

the elution of the bound IgG were performed as described

above. Samples from different stages of the purification

were quantified by Western blot using the goat antimouse

IgG-HRP conjugate and the enhanced chemiluminescence

kit (ECL) (Amersham Pharmacia Biotech, Piscataway, NJ).

For IgG purification from mouse or rabbit sera samples,

insoluble proteins were first removed by centrifugation for

5 min at 10,000g, and 0.1 mL of soluble sera fraction was

mixed with 1 mg of ELP-ProG in 0.4 mL of PBS. After

recovering the eluted IgG as described above, each sam-

ple was analyzed by silver staining. For the repeat usage

of ELP-ProG, IgG purification from rabbit serum was re-

peated three times using the same ELP-ProG sample. After

each elution step, ELP-ProG was resolubilized in cold-PBS

buffer and used for a subsequent cycle of IgG purification.

The intensity of the protein bands was quantified using a

Bio-Rad Gel Doc 2000 Gel Documentation System and the

Quantity One software.

RESULTS

Production of ELP-Protein L/G Fusion Proteins

Several Fc-binding proteins have been used as affinity li-

gands for antibody purification. The most widely used is

protein A (SpA), which binds immunoglobulin G (IgG)

from several mammalian species (Moks et al., 1986). How-

ever, the binding affinity of SpA has been reported to be

strongly dependent on pH (Lindmark et al., 1983). Protein

G and Protein L are two other affinity ligands that offer

binding to IgM, IgA, IgE, and IgD, a broader Ig sub-

class, and less pH dependence (Bjorck and Kronvall, 1984;

Akerstrom and Bjorcks, 1986; Bjorck, 1988). Because of

these benefits, proteins G and L have been used for puri-

fication of antibodies with improved binding properties

when compared with SpA (Kastern et al., 1992). In order to

provide a technology that is useful for all possible sources

of antibodies, ELP fusions containing either Protein G or

Protein L were constructed.

PCR-amplified fragments coding for Protein L and G

were fused to the 3V end of a gene coding for the ELP

domain. For a fusion containing both Protein L and G, a

fragment coding for Protein LG was amplified and fused

in the same way with the ELP domain. All fusion pro-

teins were easily produced in E. coli BL21(DE3) and

purified by two cycles of inverse temperature transition.

The purity of the fusion proteins was determined by SDS-

PAGE followed by silver staining (Fig. 1A), and bands

Figure 1. Production and purification of ELP fusion proteins. The purity of the fusion proteins was analyzed by (A) 10% SDS-PAGE followed by silver

staining, (B) Western-blot analysis with a goat IgG-alkaline phosphatase conjugate, and (C) Western-blot analysis with a human IgM-horseradish

peroxidase conjugate. G, ELP-ProG; L, ELP-ProL; LG, ELP-ProLG.

KIM ET AL.: PURIFICATION OF ANTIBODIES 375

corresponding to the expected sizes of the fusions were

observed. In the case of Protein L and Protein LG fusions,

partially degraded products were detected as observed pre-

viously with other Protein L fusions (Kihlberg et al., 1992).

Typically, 400 mg/L of ELP-ProG and 100 mg/L of ELP-

ProL/ELP-ProLG were obtained.

The presence of antibody-binding domains in the fusions

was confirmed by Western blot analysis using goat IgG-AP

(Fig. 1B) and human IgM-HRP conjugates (Fig. 1C). Con-

sistent with the binding preference, a strong interaction

was observed between Protein G and the goat IgG-AP

conjugate, while Protein L has a high affinity for human

IgM but not for the goat IgG. Neither conjugates interacted

with ELP itself, indicating the binding functionality of

the Protein L and G domains in the fusion proteins.

Characterization of Transition andAntibody-Affinity Functionalities

The transition properties of the ELP fusions were studied

by measuring the solution turbidity as a function of tem-

perature. As shown Figure 2, similar transition profiles

as ELP itself were observed for the ELP fusions, showing

that the transition property was not affected by antibody-

affinity domains.

To demonstrate the antibody-binding capability, the fu-

sion proteins were first immobilized onto a hydrophobic

polystyrene microplate by inducing aggregation at 37jC

for 30 min as described previously (Shimazu et al., 2003).

Different antibody-HRP conjugates were subsequently

added and the amount of bound antibodies was measured

by the conjugated HRP activity. As shown in Table I,

extensive binding of either goat IgG or human IgM to

the ELP fusions was observed, and the binding prefer-

ence was again consistent with the binding affinity of Pro-

tein G and Protein L. ELP without an antibody binding

domain or HRP were used as controls and virtually no

binding was observed. These results confirm that the Pro-

tein G and Protein L domains are solely responsible for

the antibody binding and even aggregated ELP fusions are

presented in an accessible orientation to interact with the

target antibodies.

IgG Purification

The IgG purification capability of the fusions was

evaluated using ELP-ProG as a model. Initial demonstra-

tions of IgG purification were conducted using purified

mouse and rabbit IgGs. After 30 min incubation at room

temperature, bound IgG was recovered by thermal pre-

cipitation. In both cases, 100% recovery of the ELP-ProG

fusion was achieved. After solubilization, the bound IgG

was eluted from the ELP-ProG-IgG complex with an ice-

cold elution buffer (pH 2.6) and the ELP-ProG fusion was

subsequently separated by thermal precipitation. All frac-

tions were analyzed by silver staining and quantified

using a Bio-Rad Gel Doc 2000 Gel Documentation Sys-

tem. As shown in Figure 3, 92% of rabbit IgG and 68% of

mouse IgG were recovered in this one-step process. The

lower recovery of mouse IgG compared to rabbit IgG is

due to the low binding affinity between Protein G and one

of mouse IgG subtype (IgG1). The reported efficiencies of

recovery are higher than those reported using Protein G

in chromatographic separations (Dancette et al., 1999;

Thomas et al., 2002).

To investigate whether the complex matrices typically

associated with IgG purification have any effect on the

recovery efficiency using the ELP fusions, the super-

natant of a hybridoma cell culture (C1B7), which pro-

duces mouse IgG against human acetylcholinesterase, was

used. By employing a similar procedure as described

above, each fraction was recovered and analyzed. Again,

100% recovery of the ELP fusions was obtained. Because

of the low concentration of IgG in the supernatant, en-

hanced chemiluminescence (ECL) was applied to quan-

tify each fraction (Fig. 4). The recovery efficiency was

Figure 2. The transition profiles of the ELP fusions. Turbidity

measurements were conducted at 620 nm from 25–40jC with 100 Al of

0.1 mM ELP or ELP fusions in PBS containing 0.5 M NaCl.

Table I. Binding of antibodies by different ELP fusion as indicated by

the conjugated HRP activity (�A490).

�A490* ELP G fusion L fusion LG fusion

Buffer 0.005 0.006 0.032 0.010

HRP 0.007 0.008 0.024 0.006

Goat IgG-HRP 0.017 1.851 0.025 0.846

Human IgM-HRP 0.031 0.046 0.927 0.842

*The amount of bound IgGs was quantified by the conjugated HRP

activity by incubating with 0.1 mL of HRP substrate for 10 min. The

absorbance was measured at 490 nm using a microplate reader.

376 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 90, NO. 3, MAY 5, 2005

calculated to be 64%, which demonstrates that the pres-

ence of cell culture supernatant has no effect on the

binding efficiency or the precipitation efficiency of the

ELP-ProG fusion.

To demonstrate the utility of the technology to purify

IgGs from animal sera, mouse and rabbit sera were exam-

ined. The serum samples were prepared by centrifugation

to remove nonsoluble proteins. Purification was performed

as before and the efficiency was determined by silver

staining (Fig. 5). Essentially a single band representing the

recovered IgG was detected in the elution fractions, while

the other serum proteins remained in the supernatant (non-

bound fractions). Parallel to the results with the purified

antibodies, around 60% of IgG in the mouse serum was

recovered, while 90% of IgG in the rabbit serum was

recovered. These results confirm that no interference

occurred during purification because of other proteins in

the sera.

In addition to the ease of purification and the high

efficiency, another significant advantage of this strategy

is the possibility of repeated usage of ELP-ProG. The

regeneration and rebinding efficiency was evaluated with

rabbit serum. The same ELP-ProG fusion was used three

times for IgG purification (Fig. 6). Each elution fraction

showed the same recovery efficiency and purity, demon-

strating that the ELP-ProG fusion can be reused for IgG

purification several times without losing binding affinity

and the inverse transition property. This result opens pos-

sible applications for ELP fusions to be useful with harsh

conditions such as low pH without losing functionality.

DISCUSSION

Affinity precipitation is a relatively new method, which

allows protein separation from cell lysates with rather high

Figure 3. Recovery of mouse (I) or rabbit (II) IgG by the temperature-triggered precipitation. A: Total IgG; B: unbound protein fraction after

precipitation; C: recovered IgG after elution; D: recovered ELP fusion after elution. Purified IgGs are indicated by an arrow.

Figure 4. IgG purification from the supernatant of hybridoma cell culture. A: supernatant of hybridoma cell culture (C1B7); B: unbound protein fraction

after precipitation; C: recovered IgG after elution; D: recovered ELP fusion after elution. Purified IgG is indicated by an arrow.

KIM ET AL.: PURIFICATION OF ANTIBODIES 377

yields compared to conventional chromatography (Gupta

and Mattiasson, 1994). Simple changes in temperature or

salt concentration also overcome the complex immobiliza-

tion and washing steps required with chromatograhic

purification. Although affinity precipitation has been

reported with PNIPAM polymers (Galaev and Mattiasson,

1993), this method requires complicated organic synthesis

as well as chemical conjugation for binding affinity.

ELP is an effective alternative to PNIPAM polymers,

offering the same reversible phase transition property over

a wide range of conditions (Urry, 1997). Unlike the sta-

tistical nature of step and chain polymerization reactions,

ELP biopolymers are specifically preprogrammed within a

synthetic gene template that can be precisely controlled

over chain length, composition and fusion partners. These

unique properties, when combined with the binding affinity

of a fusion partner, could be exploited as a powerful

method for ligand purification. We have previously dem-

onstrated this principle for protein purification based on

metal-coordinated bridging (Stiborova et al., 2003). Puri-

fication of His-tagged enzymes was achieved by coprecip-

itation with ELP through Ni2+ complexation. In this study,

we exploited antibody-binding affinity of Protein G and

Protein L in engineering ELP fusions that are useful as a

universal platform for antibody purification. The resulting

ELP fusions retained the ability to reversibly aggregate and

to bind IgGs with high affinity.

Although thermally triggered purification of proteins

based on ELP was first demonstrated by Chilkoti (Meyer

and Chilkoti, 1999; Meyer et al., 2001), fusion proteins

with ELP were used and the final recovery of purified

proteins required protease treatment. In our approach, we

have created ELP-Protein G or Protein L fusions, enabling

complexation with IgG and purification by a temperature

trigger. Subsequent separation of IgG was easily obtained

by incubating with an elution buffer. This approach is

universally applicable to all antibodies and does not re-

quire fusion construction for each individual protein or

Igs of interest.

The ELP-based method presented here is very spe-

cific, easy to manipulate, and fast, with only a few short

centrifugation steps followed by resolubilization. The sep-

aration of purified antibodies is very convenient, requiring

only a single elution step. The versatility of the method

was successfully demonstrated for the purification of anti-

bodies from different sources with similar efficiencies.

Figure 5. IgG purification from mouse (I) or rabbit serum (II). A: Total serum; B: unbound protein fraction after precipitation; C: recovered IgG after

elution; D: recovered ELP fusion after elution. Purified IgGs are indicated by an arrow.

Figure 6. Purification of IgG from rabbit serum in three (1–3) repeating

cycles. The same ELP-ProG fusion was reused for purification after IgG

elution. A: unbound protein fraction after precipitation; B: recovered IgG

after elution. Purified IgGs are indicated by an arrow.

378 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 90, NO. 3, MAY 5, 2005

Although only the results for a few subclasses of antibodies

were reported, the flexibility of creating ELP fusions with

different types of ligands can be similarly applied and

used for other antibodies. We believe that this technology

will be useful as an economical and highly efficient tool

for the purification and immobilization of antibodies.

The C1B7 hybridoma was obtained from the Developmental Studies

Hybridoma Bank developed under the auspices of the NICHD and

maintained by The University of Iowa, Department of Biological

Sciences, Iowa City, IA 52242. We thank Dr. Ulf Sjobring for

providing the plasmid coding for Protein L and G.

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