Conclusion

A generally applicable antibody purification platform was developed by using Z33-ELP for

affinity capture, precipitation and release of antibodies. Z33-ELP is a fusion protein of

ELP with a small binding domain derived from SpA. The major advantage of the small binding domain of 33 amino acids is that it can easily be fused with ELP, while binding is still dependent on the same interaction as with the complete protein. Furthermore, the Z33

Chapter 3

domain has a decreased risk of denaturation and degradation. Therefore this methodology can easily substitute the industrially-applied SpA-based affinity chromatography procedure. Z33-ELP can also be reused, since it is obtained pure after the release of the

antibodies. Even though antibodies are normally released from SpA-columns under acidic conditions, it was shown that release of antibodies from Z33-ELP could only be observed

after short treatment with base, which did not affect the functionality of the antibody. In Chapter 2, it was shown that binding of the Z33-domain to bovine IgGs was several

orders of magnitude weaker than binding to human IgGs. This selectivity towards human IgGs was employed in an efficient and selective purification of human IgGs from mixtures containing bovine IgGs and other proteins by affinity precipitation. Potentially, this means that monoclonal antibody production does not necessarily need to be performed in medium with low bovine IgG content, such as serum-free medium or medium containing fetal calf serum.[32]

3.4. Acknowledgements

We thank Rosalie Teeuwen for kindly providing the plasmid encoding for ELP[V5L2G3-

90]. Jaleesa Bresseleers is acknowledged for the work on preliminary ELP-constructs for the purification of antibodies.

3.5. Experimental section

Elastin-like polypeptide nomenclature

ELP constructs are usually described using the notation ELP[XiYjZk-n], where the capital letters between

the brackets indicate the single letter amino acid code for the residues at the Xaa position in the pentapeptide Val-Pro-Gly-Xaa-Gly. The subscript stands for the ratio of the guest residues and the n

represents the total number of pentapeptide repeats.

Construction of expression vector, and expression, purification and characterization of Z33-ELP

The construction of pET15b-Z33-ELP[V5L2G3-90] was described in Chapter 2. Protein expression in

BLR(DE3) E. coli (Novagen, MERCK), purification and characterization was performed as described in

An ELP fusion protein for selective purification of antibodies

83

and the purity was verified by SDS-PAGE. ESI-TOF (JEOL AccuTOF): Z33-ELP[V5L2G3-90] calculated

41709.2 Da, found 41708.6 Da. For control experiments, ELP[V5L2G3-90] without the Z33 domain was

expressed and purified as described in Chapter 2 using pET15b-ELP[V5L2G3-90]. The protein was

obtained in a yield of 95-120 mg/L of bacterial culture and the purity was also verified by SDS-PAGE. ESI-TOF (JEOL AccuTOF): calculated 39670.9 Da, found 39671.5 Da.

Mass spectrometry

Protein mass characterization was performed by electrospray ionization time-of-flight (ESI-TOF) on a JEOL AccuTOF. Deconvoluted mass spectra were obtained using MagTran 1.03b2. Isotopically averaged molecular weights were calculated using the “Protein Calculator v3.3” at http://www.scripps.edu/~cdputnam/protcalc.html.

Transition temperature

The transition temperatures of the ELPs were determined by turbidity measurements, where the optical absorbance at 600 nm was measured as a function of temperature (5-50 °C). These measurements were performed in a quartz cuvette with a path length of 0.1 cm on a Jasco J-815 Circular Dichroism Spectrometer equipped with a Peltier temperature controller (band width: 1 nm, response: 1 sec., sensitivity: standard, temperature slope: 1 °C/min). For these measurements, 400 µL of 1.0 mg/mL ELP in phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·2H2O, 1.0 mM

KH2PO4, pH 7.4) was prepared. For the experiment in presence of antibody, a mixture of 100 µg of Z33-

ELP (2.4 nmol) with 184 µg of FITC-labeled polyclonal rabbit anti-mouse IgGs (1.23 nmol, Dako, F0261) in 400 µL PBS was prepared.

Analysis of affinity by dot blot

The affinity of Z33-ELP for antibodies from different origins was assayed by a dot blot assay. The FITC-

labeled control IgGs and chicken IgY used in this assay were purchased from Santa Cruz, Biotechnology. Z33-ELP was applied to a nitrocellulose membrane (1 µL of 10 mg/mL). After further blocking of the

membrane with BSA (5% BSA in PBS with 0.1% Tween 20), it was incubated with 8 µg FITC-labeled antibody in 2 mL of PBST + BSA (PBS with 0.1% Tween 20 and 0.5% BSA) for two hours. The membrane was washed twice with PBST + BSA and was then analyzed on a fluorescence imager (Syngene, G:BOX Chemi-XT4, blue LEDs/SW06 filter).

Size exclusion chromatography

The SEC measurements were performed on an Amersham Ettan LC system (GE Healthcare, Diegem, Belgium) equipped with a fraction collector using a Superdex 200 PC 3.2/30 column (GE Healthcare). For each analysis, 20 µL of sample was separated on the column with a flow rate of 0.050 mL/min of PBS. Three samples were analyzed: 1) 1.9 nmol Z33-ELP, 2) 0.1 nmol polyclonal rabbit IgG (Santa Cruz,

sc-2027) and 3) a mixture of 1.9 nmol Z33-ELP and 0.1 nmol polyclonal rabbit IgG which was incubated

Chapter 3

Release conditions

The release of antibodies from Z33-ELP was assayed. For each release solution which was tested, 34.5 µg

of FITC-labeled polyclonal rabbit anti-mouse IgGs (Dako, F0261) were mixed with 100 µg of Z33-ELP in

40 µL of PBS. After 30 min incubation at 20 °C the antibody/Z33-ELP complex was precipitated and

pelleted by addition of NaCl upto 1.7 M (from 5 M NaCl stock solution) and centrifugation (15000 g for 15 min at 20 °C). The supernatant was removed and the pellet was resuspended in 20 µL of the release solution. After 5 min of incubation at 20 °C, Z33-ELP was precipitated and pelleted by addition of NaCl

upto 2.5 M (from 5M NaCl stock solution) and centrifugation (15000 g for 10 min at 20 °C). The supernatant was collected and 20 µL of PBS was added. Then the pellet was resuspended in 60 µL of PBS. For each release condition, an equal fraction of the supernatant and the pellet were loaded on an SDS-PAGE gel, which was then analyzed on a fluorescence imager (Syngene, G:BOX Chemi-XT4, blue LEDs/SW06 filter). ImageJ 1.38x was used to quantify the amount of antibody present in each fraction.

Antibody capture, precipitation and release

FITC-labeled polyclonal rabbit anti-mouse IgGs (Dako, F0261) were incubated with purified Z33-

ELP[V5L2G3-90] for 30 min at room temperature in the PBS. After incubation, NaCl was added to a final

concentration of 1.7 M (from 5 M NaCl stock solution) and the mixture was centrifuged (15000 g for 10 min at 20 °C). The pellet was resuspended in 50 mM NaOH and the supernatant was stored for SDS- PAGE analysis. After 5 min of incubation at 20 °C the antibody/Z33-ELP complex was precipitated and

pelleted by addition of NaCl upto 2.5 M (from 5M NaCl stock solution) and centrifugation (15000 g for 10 min at 20 °C). The supernatant was collected and neutralized by addition of HCl and PBS. Then the pellet was resuspended in PBS. For analysis, an equal fraction of the wash, elution and pellet fraction was loaded on an SDS-PAGE gel, which was then analyzed on a fluorescence imager (Syngene, G:BOX Chemi-XT4, blue LEDs/SW06 filter). ImageJ 1.38x was used to quantify the amount of antibody present in each fraction. The activity of the purified rabbit anti-mouse IgGs was then assayed by a dot blot in which the monoclonal mouse anti αB crystallin IgGs were immobilized on a nitrocellulose membrane. The dot blot was performed as described previously. As a negative control, the same procedure was performed with ELP[V5L2G3-90].

To investigate the purification of antibodies from mixtures, the polyclonal FITC-labeled rabbit anti- mouse IgGs (Dako, F0261) were mixed with four commercially available proteins. The following mixture was prepared: polyclonal FITC-labeled rabbit anti-mouse IgGs (1.7 mg/mL), bovine serum albumin (BSA, 66 kDa, 3.5 mg/mL), horseradish peroxidase (HRP, 40 kDa, 1.0 mg/mL), Candida antarctica lipase

B (CalB, 34 kDa, 0.7 mg/mL) and lysozyme (14 kDa, 1.6 mg/mL) in PBS. Then a similar purification procedure as described above was employed. However, an additional washing step with PBS was performed prior to elution to completely remove the contaminants.

Recycling of Z33-ELP

For the recycling experiments, the purification of FITC-labeled polyclonal rabbit anti-mouse IgGs (Dako, F0261, 23 µg, 0.15 nmol) was repeated three times. After the elution of the antibody, the pelleted Z33-

An ELP fusion protein for selective purification of antibodies

85

an equal fraction of the three wash fractions, the three elution fractions and the final pellet fraction was loaded on an SDS-PAGE gel, which was then analyzed on a fluorescence imager (Syngene, G:BOX Chemi-XT4, blue LEDs/SW06 filter). ImageJ 1.38x was used to quantify the amount of antibody present in each fraction.

Selective purification of human antibodies

A stock solution containing ELP (125 µM) and Z33-ELP (12.5 µM) was prepared. Human IgG1 (Sigma-

Aldrich, I5029, 12 µg, 0.08 nmol) was mixed with a solution of FITC-labeled bovine IgG (12 µg, 0.08 nmol) and BSA in PBS (Santa Cruz Biotechnology, sc-2719) to yield a solution with a total volume of 40 µL. To this mixture, 10 µL of the ELP/Z33-ELP stock solution was added (final quantities: ELP: 50 µg,

1.25 nmol, Z33-ELP: 5 µg, 0.12 nmol). Then the Z33-ELP was allowed to bind to the IgGs during

incubation for 20 min at room temperature. After this incubation, NaCl was added to a final concentration of 1.7 M (from 5 M NaCl stock solution) and the mixture was centrifuged (15000 g for 5 min at 20 °C). To remove the non-specifically bound proteins, the pellet was redissolved in 50 µL PBS, and the supernatant was stored for SDS-PAGE analysis (wash fraction 1, 2 and 3). Then the antibody- Z33-ELP complex was again precipitated by addition of NaCl to a final concentration of 1.7 M (from 5 M

NaCl stock solution) and the mixture was centrifuged (15000 g for 5 min at 20 °C). This washing was repeated two more times and eventually the pellet was redissolved in 50 mM NaOH to release the antibody from Z33-ELP. After 5 min of incubation at 20 °C, Z33-ELP was precipitated and pelleted by

addition of NaCl up to 2.5 M (from 5 M NaCl stock solution) and centrifugation (15000 g for 5 min at 20 °C). The supernatant was collected and neutralized by addition of HCl and PBS (elution fraction). Then the pellet was redissolved in PBS. For analysis, the three wash fractions, the elution fraction and the pellet fraction were loaded on an SDS-PAGE gel, which was then analyzed on a fluorescence imager (Syngene, G:BOX Chemi-XT4, blue LEDs/SW06 filter). ImageJ 1.38x was used to quantify the amount of antibody present in each fraction. This quantification was corrected for differences in loading volumes of the various fractions.

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An ELP fusion protein for selective purification of antibodies

Synthesis and self-assembly of elastin-like