Reverse phase chromatography

To further purify the Pegasus DZF peptide, RP chromatography (using HPLC) was applied.

Using this approach, peptides are separated based on their “hydrophobic character”. Peptides adsorb to the hydrophobic nonpolar surface of the column after applying them in a polar mobile phase by interaction of the nonpolar components of the proteins with the hydrophobic nonpolar stationary phase. Compounds are eluted from RP HPLC columns by decreasing the polarity of the mobile phase using organic solvents which results in desorption of the protein.

To assure that separation is solely based on hydrophobicity, the effective charges of the peptide have to be reduced before loading it onto the column. This can be accomplished by reducing the pH (typically to 2) and at the same time providing an ion-pairing reagent (e.g.

TFA) that “hides” the resulting positive charges by forming “ion pairs” with them.

Thus, the sample was acidified with 10 % TFA to pH 2-3 to provide binding to the hydrophobic stationary phase of the C4 reverse phase column that is solely based on the hydrophobic character of the peptide. The protein was then loaded onto the column using nonpolar buffer conditions (Buffer A, consisting of water with 0.1 % TFA). Molecules were eluted differentially with an organic mobile phase (Buffer B, containing acetonitrile with 0.1

% TFA) that was introduced gradually. As shown in Figure 5.5A, three major peaks eluted at different concentrations of buffer B. Several fractions together with the peaks were captured and analyzed by SDS-PAGE and subsequent silver staining demonstrating that the Pegasus DZF domain eluted as a single peak between 68-71 % of buffer B (Figure 5.5B). In addition, the captured sample was highly pure and contained only the Pegasus DZF domain. (Note that the other peaks did not contain any proteins and are likely to represent buffer and DTT eluting from the column). The same buffer conditions were then used to scale up the sample amount using a shallower gradient for eluting the peptide. As expected, this chromatography run yielded more protein as judged by the absorbance units and the captured volume (data not shown). The HPLC peak fraction corresponding to the Pegasus DZF peptide was subsequently lyophilized and the dried product was moved to an anaerobic chamber.

5.2.5 Refolding

After these successful purification steps, we attempted to refold the Pegasus peptide with either cobalt or zinc. To do this, the dried peptide was resuspended in water (in an anaerobic chamber) and the protein concentration was determined (~ 25 mg/ml in this initial experiment). To attempt refolding the peptide in its active form, 1.5 molar equivalents of cobalt were added and the pH was adjusted by introducing different buffers. The solution of dissolved Pegasus peptide immediately turned blue after adding the buffer, suggesting that the peptide was coordinating the cobalt ion (data not shown). However, insoluble aggregates accumulated immediately following appearance of the blue color. Spinning down the sample demonstrated that the aggregates represented the refolded peptide since the precipitate was blue and the supernatant did not harbor any detectable protein as judged by UV absorbance

1 2 3 A B 4 5 6 7 8 9 C

A B C

10 kD

A

B

1 2 3 A B 4 5 6 7 8 9 C

A B C

10 kD

1 2 3 A B 4 5 6 7 8 9 C

1 2 3 A B 4 5 6 7 8 9 C

A B C

A B C

10 kD 10 kD

A

B

Figure 5.5 Analysis of the Reverse Phase (RP) chromatography run applied to further purify the Pegasus DZF. (A) Analytical HPLC trace for the DZF domain from Pegasus. Bound peptides were eluted over ~ 60 min using a Buffer B gradient (shown in turquoise). UV trace is shown in purple. Collected peaks are numbered A, B, C. (B) SDS-PAGE analysis of the HPLC run from (A). Peak fractions A-C together with several fractions (2-9) taken at different time points were collected and analyzed. Lane 1 represents a sample of the acidified peptide before it was loaded onto the column. The red arrow indicates the Pegasus DZF domain band and the black arrow indicates where the 10 kD band of the Standard ran.

measurement. Using zinc instead of cobalt or performing the folding reaction on ice also resulted in a precipitation of protein following adjustment of the pH.

The efficiency of refolding depends in general on the competition between correct folding events and aggregation (reviewed in Lilie et al., 1998). Thus, it is very important to slow down the aggregation process in order to obtain correct folded and biological active protein.

There a various biochemical variables that influence the formation of properly folded protein which were gradually evaluated as described below.

5.2.5.1 Protein concentration

A very direct way of minimizing aggregation is by reducing the concentration of the protein, since aggregation usually happens at high concentrations of proteins (reviewed in Clark, 1998). Thus, the protein concentration of the Pegasus DZF peptide was decreased stepwise by serially diluting purified unfolded peptide with water. The following final concentrations of protein were tested in this experiment: 10, 0.1, 0.05, 0.001 and 0.0001 mg/ml. We attempted to re-fold these diluted peptides using the method described above and the color change was monitored. The result of this experiment showed that reduction of the protein concentration still led to formation of precipitates. Going below a certain protein concentration (< 0.001 mg/ml) did not permit detectable levels of blue color anymore (as determined by eye).

5.2.5.2 Folding buffer composition (pH, ionic strength)

Other variables that influence the stability of the proper folded state are the pH and the ionic strength of the folding buffer (reviewed in Clark, 1998; reviewed in Lilie et al., 1998). Thus, different buffer conditions (pH 5.0 to pH 8.0) were used and salt was added at various concentrations to refold the protein. As shown in Table 5.2, the peptide folded but precipitated at pH 7.0 - 8.0 but then was not able to fold at pH 5.0 or pH 6.0 as judged by the color change or lack thereof. Adding salt at different concentrations did not prevent precipitation once the protein was folded (Table 5.3). Introducing salt to the folding reaction before adding cobalt resulted in an immediate precipitation and the color did not change, even after introducing cobalt.

Buffer, pH Folding phenotype 0.5 M MES, pH 5.0 No change in color. No precipitation 0.5 M MES, pH 6.0 No change in color. No precipitation 1 M HEPES, pH 7.0 Blue precipitate

1 M BTP, pH 7.0 Blue precipitate 1 M HEPES, pH 7.5 Blue precipitate 1 M BTP, pH 7.5 Blue precipitate 1 M HEPES, pH 8.0 Blue precipitate 1 M BTP, pH 8.0 Blue precipitate

Table 5.2 Evaluation of different folding buffers with different pH values used to perform the refolding reaction.

5.2.5.3 Urea

Urea has been proven to inhibit aggregation by increasing the solubility of unfolded proteins and decreasing non-specific hydrophobic interactions which results in a general increase of correctly folded protein (Orsini and Goldberg, 1978). Thus, urea was added at various concentrations (0-2 M) to refold the protein either in combination with salt or without adding salt. As shown in Table 5.3, even after introduction of urea we still observed blue precipitates.

Buffer Salt Urea Folding phenotype

0 M 0 mM NaCl, KCl or NH₄OAc 1 M

2 M

Blue precipitate 0 M

10 mM NaCl, KCl or NH₄OAc 1 M

2 M

Blue precipitate 0 M

1 M 1 M HEPES,

pH 7.5

200 mM NaCl, KCl or NH4OAc

2 M

Precipitation before adding cobalt, no color change after

adding cobalt

Table 5.3 Evaluation of different folding buffer compositions (pH, ionic strength and addition of urea) used to perform the refolding reaction. 1 M HEPES, pH 7.5 was used for all reactions. Three different salts at three different concentrations (column 2) were tested either in combination with urea (used in two concentrations) or without adding urea.

5.2.5.4 Additives

It has been shown that the use of refolding additives can prevent aggregation by interfering with intermolecular hydrophobic interactions. A variety of additives have been described that prevent aggregation by stabilizing the proper folded state, by destabilizing incorrect folded

peptides, and by enhancing the solubility of either folding intermediates or unfolded peptides.

Examples for such additives are detergents, surfactants and sugars which have proven to minimize aggregation and increase the yield of properly folded protein (Maeda et al., 1996;

reviewed in Clark, 1998). Thus, we tested a large series of different additives which were introduced to the folding reaction in order to prevent aggregation. To do this, a commercially available detergent screen kit was used which provided 72 unique detergents (Hampton Research, detergent screen 1, 2 and 3). None of the provided detergents was able to prevent aggregation during the refolding step (data not shown).

Since we were not able to find proper folding conditions for the Pegasus DZF peptide, we decided to purify the DZF domain from human Ikaros (work performed by R. Fang), human TRPS-1, Hunchback D.m. and Hunchback C.e., as well as the synthetic DZF domain Tr-Eo-Eo applying the same purification strategy used for the Pegasus DZF domain. The hope was that using different proteins might help to solve the aggregation problems since the aggregation could be due to various surface-exposed hydrophobic amino acids in the Pegasus peptide. Another reason for the aggregation could be the formation of higher order oligomers as described for Eos (Westman et al., 2003) and Ikaros (McCarty et al., 2003) which in turn could result in a precipitation of the proteins. Thus, the DZF domain from Hunchback C.e.

and the synthetic Tr-Eo-Eo domain were chosen because they do not mediate homodimerization at all or only weakly, respectively, as judged by the B2H (see Chapter 3, sections 3.2.4 and 3.4.1) and B1H system (data not shown).