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Increased adsorption of histidine-tagged proteins onto tissue culture polystyrene
Colloids and Surfaces B: Biointerfaces 92 (2012) 286–292
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Increased adsorption of histidine-tagged proteins onto tissue culture polystyrene Maria Holmberg 1 , Thomas Steen Hansen 2 , Johan Ulrik Lind, Gertrud Malene Hjortø ∗ Department of Micro- and Nanotechnology, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark
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Article history: Received 13 October 2011 Received in revised form 2 December 2011 Accepted 2 December 2011 Available online 16 December 2011 Keywords: Histidine-tagged protein Adsorption Tissue culture polystyrene Electrostatic interaction Blocking molecule
a b s t r a c t In this study we compare histidine-tagged and native proteins with regards to adsorption properties. We observe significantly increased adsorption of proteins with an incorporated polyhistidine amino acid motif (HIS-tag) onto tissue culture polystyrene (TCPS) compared to similar proteins without a HIS-tag. The effect is not observed on polystyrene (PS). Adsorption experiments have been performed at physiological pH (7.4) and the effect was only observed for the investigated proteins that have pI values below or around 7.4. Competitive adsorption experiments with imidazole and ethylenediaminetetraacetic acid (EDTA), as well as adsorption performed at different pH and ionic strength indicates that the high adsorption is caused by electrostatic interaction between negatively charged carboxylate groups on the TCPS surface and positively charged histidine residues in the proteins. Pre-adsorption of bovine serum albumin (BSA) does not decrease the adsorption of HIS-tagged proteins onto TCPS. Our findings identify a potential problem in using HIS-tagged signalling molecule in assays with cells cultured on TCPS, since the concentration of the molecule in solution might be affected and this could critically influence the assay outcome. © 2011 Elsevier B.V. All rights reserved.
1. Introduction One of the most common procedures for purification of recombinant proteins is to incorporate a so-called histidine-tag (HIS-tag) in the protein amino acid sequence [1–3]. Such a HIS-tag typically consists of six histidine residues placed in either the N- or C-terminus of the protein. Histidine tagging enables efficient protein purification on an affinity column by chelation between the histidine residues and fixed transition metals such as Cu(II) and Ni(II) [4,5]. This method for purification of proteins is popular and many commercially available products have a polyhistidine amino acid motif incorporated into the protein. However, a number of reports have emerged regarding how the incorporation of a HIS-tag can result in unexpected side-effect such as the formation of protein hexamers [6], altered levels of catalytic activity in integrases [7], increased phosphorylation of proteins and subsequently resistance to thrombin cleavage [8], conformational changes [9] and assembly of proteins into sheet structures [10].
∗ Corresponding author. Tel.: +45 45 25 81 56. E-mail address: [email protected] (G.M. Hjortø). 1 Present address: Danish Technological Institute, Gregersensvej, Taastrup, Denmark. 2 Present address: Radiometer Medical ApS, Åkandevej, Brønshøj, Denmark. 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.12.001
In the present study we report another side-effect, namely that the incorporation of a HIS-tag in proteins can lead to significantly increased adsorption of the protein onto TCPS surfaces, compared to the native protein. TCPS is used in many different setups for cell culturing and is normally considered to be a standard cell compatible material [11,12]. An unexpected high adsorption of HIS-tagged proteins onto TCPS could potentially deprive the protein concentration in solution along with increasing the protein concentration at the surface. Such adsorption of proteins to the TCPS surface can thus have a large effect on the outcome of an assay, especially in biological assays where the effective concentrations of relatively small proteins, like growth factors and chemokines, most often lie in the range of a few ng/ml. The increased adsorption of HIS-tagged proteins was not seen on regular PS surfaces, indicating that hydrophobic interaction does not play a significant role in the observed phenomenon. In contrast to the rather hydrophobic and chemically inert PS surface, the TCPS surface contains a variety of functional groups, including carboxylate groups that are introduced to the surface by corona-discharge [13], making the surface hydrophilic. The increased adsorption of the HIS-tagged proteins onto the TCPS surface appears to be related to direct interactions between histidine residues in the HIS-tag and carboxyl acid groups on the TCPS surface, as adding imidazole or EDTA to the solutions results in decreased adsorption of HIS-tagged proteins. This direct interaction appears to be mainly of electrostatic character, between protonated histidine residues and de-protonated carboxylic acids. This was indicated by a notably
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decreased adsorption of HIS-tagged proteins when changing pH outside the physiologic range or when increasing the ionic strength of the buffer. The increased adsorption of HIS-tagged proteins onto TCPS was observed for proteins with a pI below 7.4. We suggest that for native proteins with pI below physiological pH, and thus with an overall negative surface charge at pH 7.4, the possibility for strong binding through electrostatic interaction between native protein and the negatively charged TCPS surface is low. The introduction of a HIS-tag in the protein makes a stronger interaction between the protein and the surface possible. In the case of a protein with pI above 7.4 and thus with an overall positive charge at physiological pH, even the native protein has the possibility for adsorbing to the TCPS surface through electrostatic interactions. For these proteins we do observe a slightly smaller adsorption of the HIS-tagged version, which might suggest that the overall positive surface charge at physiological pH either overshadow and/or disrupt interactions between HIS-tag and carboxyl acid groups on the TCPS surface. However, other factors such as protein conformation, protein size, and how the proteins interact with BSA carrier proteins can have an impact on adsorption characteristics of the proteins. In this study we have investigated the adsorption behaviour of a set of proteins, with and without HIS-tag, onto two different polymer surfaces, TCPS and PS. We show that adsorption of HIStagged proteins with a pI below 7.4 is much higher than adsorption of corresponding native proteins at pH 7.4 onto TCPS. We further investigate how adsorption is influenced by changing parameters such as pH, blocking procedure and ionic strength in buffer solution, using the independent techniques ELISA and radioactive labelling.
2. Materials and methods Proteins were purchased from R&D Systems (R&D Systems, USA), Abcam (Abcam, UK), Invitrogen (Invitrogen, USA), Sigma–Aldrich (Sigma–Aldrich, Germany) and BioVision (BioVision, USA) as presented in Table 1, also listing the theoretical values of molecular weight (MW) and isoelectric point (pI) of the proteins. Values for proteins used in both native and HIS-tagged form were obtained using the ‘ProtParam’ software available at www.expasy.com while values for BSA, Immunoglobulin G (IgG) and fibrinogen (Fg) are citations from literature [14,15]. Phosphate buffered saline (PBS) from Lonza (Lonza Copenhagen ApS, Denmark) with 1 mg/ml BSA (Sigma–Aldrich, Germany) was used for making protein solutions. IgG and Fg were derived from human plasma (Sigma–Aldrich, Germany). Goat anti-human IL4 antibody was from R&D Systems (cat. no. AF-204-NA), Rabbit anti-human VEGF antibody was from Abcam (cat. no. ab46154), Rabbit antiStreptavidin antibody was from Abcam (cat. no. ab59320). Rabbit anti-Goat IgG – HRP (H+L) was from Invitrogen (cat. no. 611620) and Goat anti-Rabbit IgG (H+L) was from KPL (cat. no. 474-1506). For ELISA experiments TCPS and PS 96 well ELISA plates from NUNC (Thermo Fisher Scientific, Denmark) was used. ELISA adsorption experiments were carried out using protein solutions with 1 mg/ml BSA as carrier protein. Experiments were performed in quadruplicates and at least two independent experiments were performed for each protein. The results presented are average values from one experiment with four replicates, where the error bars represents the standard deviation of the mean. TCPS and PS ELISA plate wells were incubated with BSA solution (200 l, 1 mg/ml) for 30 min. Subsequently the plates were washed three times in PBS buffer containing 0.1% Tween 20 and finally once with MilliQ water (this general washing procedure was used in all further washes). Protein adsorption, either with native or HIS-tagged protein, was carried out for 30 min (100 l, 1 g/ml unless otherwise stated). The surfaces were washed again
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as described above. Primary antibodies were added to the wells (100 l, 1 g/ml) and the reaction between adsorbed protein and antibody was allowed to proceed for 30 min before the surfaces were washed again. For ELISA with PG and PG-HIS, no primary antibody was used since the conjugated antibody can bind directly to Protein G molecules. The reaction with HRP-conjugated antibody (IgG-HRP) was carried out for 30 min (100 l, concentrations as recommended by suppliers). The plates were washed and allowed to react with 50 l TMB (3,3 ,5,5 -tetramethylbenzidine) (Sigma–Aldrich) for 3 min (if not otherwise stated). The enzymatic reaction was stopped by adding 0.2 M sulphuric acid. The colour development in each well was detected using an ELISA plate reader (VICTOR, Perkin Elmer, USA) measuring the absorbance at 450 nm for 1 s in each well (CW lamp filter P450). The TCPS and PS plates (96 well) used in radioactive labelling experiments were the same kind as those used in ELISA experiments. The Iodo-Gen method (Perkin-Elmer Life and Analytical Science, USA) was used to radiolabel the proteins with radioactive iodine, where 0.2 ml protein was mixed with 0.02 ml radioiodine and kept in the Iodo-Gen tube for 20 min before the solution was removed. PG and PG-HIS was labelled with 131 I and BSA was labelled with 125 I. The labelled proteins were separated from free iodine and other chemical reagents using gel chromatography (PD10 desalting column, Amersham Bioscience, UK). Concentrations of labelled proteins were calculated using the amount of added protein and volume of final solution, typically resulting in a concentration of a couple of hundreds of g/ml. Loss of protein during separation was approximated to be around 20%. The amount of free radioiodine in solutions with labelled protein was determined using TCA (trichloroacetic acid) precipitation, revealing the free radioiodine in solution to be around 5%. Adsorption onto blocked and unblocked surfaces was performed at 20 ◦ C and labelled BSA was used as carrier protein. For blocked surfaces, 200 l of 1 mg/ml BSA was added to the wells of a 96 well ELISA plate and allowed to adsorb for 30 min before washing (the same washing procedure as that used during ELISA experiments). 100 l of 1 g/ml PG or PG-HIS was added to the wells and allowed to adsorb for 30 min before the surfaces were washed again. Each substrate was placed in a clean plastic bag and radioactivity was detected using a gamma counter (Canberra Gamma Counter, USA). The radioactivity of 125 I and 131 I on each surface was measured by counting the 27–36 keV gamma rays of 125 I and the 364 keV gamma ray of 131 I. The contribution of 131 I isotope to the counting area of 125 I has been subtracted. The counting efficiency of the detector for 131 I (<5%) is lower than for 125 I (∼50%), and contribution of 131 I to the total 125 I counts in the 26–37 keV energy range is normally lower than 10%. By counting radioactivity of 125 I and 131 I in standard solutions (Ast ) with known amount of protein (mst ), the detected radioactivity on sample surface (As ) can be transformed to amount protein adsorbed (ms ) by using the formula; ms = (mst /Ast ) × As . Each adsorption experiment was performed in triplicates and the results presented are average values of measurements on three samples, with the error bars representing the standard deviation of the mean.
3. Results and discussion Fig. 1 displays results from ELISA analysis on adsorption of (a) PG, (b) SA, (c) VEGF and (d) IL4, all in a native and a HIS-tagged version, onto PS and TCPS. PG-HIS, SA-HIS and VEGF-HIS adsorb to TCPS to a significantly higher degree than the untagged versions of the same proteins. This effect is not seen in the case of the PS surface, where the proteins adsorb at a comparable and lower level, regardless of the presence or absence of HIS-tag in the proteins. In the case of IL4, the untagged protein adsorbs to a higher degree than the untagged
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Table 1 Abbreviations, supplier and theoretical values of proteins used in the study. The theoretical values of protein that were used in both native and HIS tagged form were evaluated using the ‘ProtParam’ software available at: www.expasy.org, using the amino acid sequence of the supplier. Theoretical values of BSA, IgG and Fg are based on references as indicated in the table. Protein
Interleukin-4 Interleukin-4 with a HIS-tag Vascular endothelial growth factor Vascular endothelial growth factor with a HIS-tag Streptavidin Streptavidin with a HIS-tag Protein G Protein G with a HIS-tag Bovine serum albumin Immunoglobulin G Fibrinogen
Abbreviation
IL4 IL4-HIS VEGF VEGF-HIS SA SA-HIS PG PG-HIS BSA IgG Fg
onto both surfaces, and both IL4 and IL4-HIS adsorb more to TCPS than to PS. The higher adsorption of HIS-tagged proteins onto TCPS compared to proteins without a HIS-tag was also investigated using radioactive labelled proteins, as shown in Fig. 2. PG and PG-HIS was labelled with radioactive iodine and adsorbed onto TCPS and PS using a similar protocol as that used during ELISA experiments (see Section 2). The radioactivity on the samples was detected and
Theoretical values
Supplier (cat. no.)
MW (Da)
pI
14963.2 15,786 19094.8 19917.6 65963.6 69254.8 21131.2 21,954 67,000 [15] 150,000 [15] 340,000 [15]
9.26 9.26 7.75 7.63 6.1 6.77 4.48 4.81 4.8 [14] 6.4–7.2 [14] 5.5 [14]
R&D (204-IL) Abcam (ab53342) R&D (293-VE) Abcam (ab55566) Invitrogen (S888) Abcam (78833) Sigma–Aldrich (194559) Biovision (6510-5) Sigma–Aldrich (85040C) Sigma–Aldrich (I4506) Sigma–Aldrich (F4883)
the corresponding amount of adsorbed protein on each surface was calculated. This experiment confirmed that HIS-tagged PG adsorbs to TCPS to a significantly higher degree than the untagged version of the same protein and that both proteins (PG and PG-HIS) show similar low adsorption onto PS. Data presented in Figs. 1 and 2, indicate that the HIS-tagged versions of PG and SA have a higher affinity for the negatively charged, hydrophilic TCPS surface, than for the PS surface. PG and SA have
Fig. 1. Adsorption of (a) PG and PG-HIS, (b) SA and SA-HIS, (c) VEGF and VEGF-HIS, and (d) IL4 and IL4-HIS onto TCPS and PS. PG-HIS, SA-HIS, and VEGF-HIS show higher adsorption than PG, SA and VEGF onto TCPS, while the effect is not observed on PS. In the case of IL4, the protein without a HIS-tag shows higher adsorption than the protein with a HIS-tag on both TCPS and PS. The presented data are normalized signals from the VICTOR ELISA plate reader (Perkin Elmer, USA) and there is a linear correlation between the signal and the amount of adsorbed protein. Surfaces were blocked with 1 mg/ml BSA for 30 min before adsorption, and 1 mg/ml BSA was used as carrier protein during adsorption of 1 g/ml of investigated protein (native and HIS-tagged) (30 min). Afterwards the samples were washed and ELISA was performed according to the protocol described in Section 2.
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Fig. 2. Adsorption of PG and PG-HIS onto TCPS and PS detected using radioactive labelling, showing a significantly higher adsorption of PG-HIS than PG onto TCPS while both proteins show similar and lower level of adsorption onto PS. Surfaces were blocked with 1 mg/ml BSA for 30 min before adsorption, and 1 mg/ml BSA was used as carrier protein during adsorption of 1 g/ml PG and PG-HIS (30 min).
isoelectric points below 7.4 (see Table 1), the pH at which the experiments are carried out, and thus have an overall negative charge during adsorption. We suggest that when introducing a HIS-tag in the proteins, a local concentration of positive charges that can take part in electrostatic interactions with the carboxylate groups on the TCPS surface is added to the proteins, and that this is the principle cause of the increased adsorption, seen for these HIS-tagged proteins. VEGF shows a similar adsorption pattern as PG and SA, with higher adsorption of VEGF-HIS than VEGF on TCPS and the same level of adsorption of the two proteins on the PS surface. However, the difference in adsorption between VEGF and VEGF-HIS onto TCPS is smaller compared to what is observed for PG and SA. VEGF has a theoretical isoelectric point just above 7.4 (see Table 1) and can be expected to be overall neutral, with tendency to have a small positive charge, at physiological pH. This might explain the higher adsorption of the native protein and the less pronounced effect of the addition of a HIS-tag to VEGF regarding the adsorption onto TCPS. For IL4, with a pI well above 7.4 and an overall positive charge at the experimental conditions used, adding a HIS-tag to the protein does not necessarily improve the interaction with the TCPS surface. Both IL4 and IL4-HIS can thus be expected to show a rather high adsorption onto TCPS caused by electrostatic interaction between protein and surface.
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To investigate if the increased binding of HIS-tagged proteins onto TCPS was caused by direct interaction between the HIS-tag and the TCPS substrate, experiments with competitive adsorption were performed. Fig. 3 presents the adsorption of 10 ng/ml PG-HIS onto TCPS in competition with (a) imidazole and (b) EDTA. Imidazole is an organic compound with molecular weight of approximately 68 Da,which is identical to the ring structure present in the histidine side chain. EDTA is a poly-acid containing four carboxylate groups, and a strong chelator of divalent cations. The latter should be a negligible effect in our studies as buffers without Mg2+ and Ca2+ were generally used. From the above, one can expect that imidazole can compete with the HIS-tag in the modified proteins for interacting with the TCPS surface, while EDTA can compete with the carboxylate groups on the TCPS surface for interacting with the HIS-tag. As observed in Fig. 3, adsorption of PG-HIS onto TCPS decreases with increased concentration of both imidazole and EDTA. All EDTA concentrations tested resulted in a significant reduction of protein adsorption. At EDTA concentrations below 10 mM, the reduction in protein adsorption is probably due to competition between the EDTA groups and the carboxylates on the surface for interaction with the HIS-tag on the proteins. A further drop, as that observed for 100 mM EDTA could also be partly an effect of the increased ionic strength of the solution achieved by the high concentration of EDTA molecules present at this concentration. The effect of adding higher concentrations of imidazole strongly suggest that the histidine residues in the proteins are involved in the higher adsorption of the HIS-tagged proteins. The suggestion that the higher adsorption is caused by electrostatic interaction between histidine residues and carboxylate groups was further investigated looking at adsorption of HIS-tagged proteins as a function of pH and ionic strength in the buffer. A change in pH will result in changes in the level of protonation of carboxylate groups on the TCPS surface and of the histidine residues in the protein, while an increase in the ionic strength will shield electrostatic interactions in general. In Fig. 4, adsorption of 100 ng/ml PG and PG-HIS onto TCPS is shown as a function of pH (pH 4–9). Reaction with TMB during ELISA experiments was carried out approximately 6½ times longer for PG than for PG-HIS. The longer reaction time for PG was necessary to obtain a strong enough absorbance signal for ELISA reader detection. The weak signal was due to the much lower adsorption of native PG compared to PG-HIS. As observed in Fig. 4, an overall higher adsorption of PG-HIS compared to PG onto TCPS was observed at all pH values. Furthermore, there was a shift of maximum adsorption to a higher pH value for PG-HIS compared to PG. The carboxylate groups on the TCPS
Fig. 3. Adsorption of PG-HIS onto TCPS in the presence of different concentrations of (a) imidazole and (b) EDTA showing the normalized signal from the ELISA plate reader for which there is a linear correlation between the signal and the amount of adsorbed protein. The adsorption of PG-HIS decreases with increased concentration of both imidazole and EDTA, indicating that the high adsorption of PG-HIS relies on interaction between the histidine residues in the protein and the carboxylate groups on the TCPS surface. Surfaces were blocked with 1 mg/ml BSA for 30 min before solutions of 10 ng/ml HIS-tagged protein, carrier protein (1 mg/ml BSA) and either imidazole or EDTA were added to the surface (30 min). Afterwards the samples were washed and ELISA was performed according to the protocol described in Section 2.
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Fig. 4. Adsorption of 100 ng/ml PG and PG-HIS onto TCPS as a function of pH. The adsorption of PG-HIS is generally higher than the adsorption of PG and the maximum adsorption for PG-HIS is positioned at a higher pH than the maximum value of PG. The signal from the ELISA reader, which is linearly correlated with the amount protein adsorbed, has been normalized. It should be noted that for obtaining a detectable signal from PG adsorption, the TMB reaction step had to be carried out for a time period that was more than 6 times longer than that for PG-HIS adsorption. Otherwise the adsorption protocol for PG and PG-HIS was identical and 30 min of 1 mg/ml BSA blocking was performed before 30 min of adsorption of PG or PG-HIS with BSA (1 mg/ml) as carrier protein was carried out.
surface can be expected to have a pKa around 5 and HuyghuesDespointes et al. [16] report that the pKa of histidine is 6.5–7.0 when forming ionic pairs with a carboxylic acid (aspartate). Thus, at pH below 5 the carboxylic acids are increasingly protonated and at pH above 7.0 the histidine residues are de-protonated. This correlates well with the drop in adsorption of PG-HIS onto TCPS below pH 5 and above pH 7.4 strengthening the hypothesis that the high adsorption is caused by electrostatic interaction between carboxylate groups on the TCPS surface and histidine residues in the protein. The shift of maximum adsorption to a higher pH value for PG-HIS can be expected by a shift to a higher pI for the HIS-tagged protein compared to the protein without a HIS-tag (see Table 1). The electrostatic attraction between the carboxylic acid and HIStag is expected to be inversely proportional to the ionic strength of the solution due to increased shielding [17]. Experiments were conducted with varying ionic strength to verify that electrostatic attraction is the main mechanism in the observed adsorption of histidine-tagged proteins. Fig. 5 shows adsorption of 100 ng/ml PG-HIS onto TCPS as a function of NaCl concentration (0–800 mM) in the buffer. Here, a dramatic decrease in adsorption is observed when the NaCl concentration increases from 0 mM to 800 mM. The experiments with pH and ionic strength support that the main mechanism responsible for the increased adsorption of HIS-tagged proteins is electrostatic attraction. BSA blocking is a universal method for decreasing unspecific adsorption of proteins to surfaces. The experimental conditions used in this study are similar to conditions used within ELISA, as well as many other biological assays where BSA blocking is employed. Thus it is relevant to consider the influence of BSA blocking on the observed adsorption patterns. In all experiments performed, BSA has been used as a carrier protein and is thus present in a much higher concentration (mg/ml) than the proteins investigated for unspecific adsorption onto PS and TCPS (ng-g/ml). Furthermore, in all ELISA experiments presented surfaces have been blocked with 1 mg/ml BSA for 30 min before performing adsorption with the proteins that are under investigation. As electrostatic interactions are short range in aqueous environments, the proteins need to be in physical contact with the surface for these interactions to come into play, and establish a binding
Fig. 5. Adsorption of PG-HIS onto TCPS as a function of NaCl concentration in the buffer showing a decrease in adsorption with increased ionic strength. The signal from the ELISA reader correlates linearly with amount protein adsorbed on the surface and has been normalized. The surface has been blocked with 1 mg/ml BSA for 30 min before adsorption with 100 ng/ml PG-HIS in PBS buffer (with 1 mg/ml BSA as carrier proteins) with different NaCl concentrations was carried out.
that withstands the rinsing procedures used in this study. The high level of HIS-tagged proteins on TCPS thus indicates that there has to be exposed areas of the polymer surface during the incubation. The possibility for HIS-tagged proteins to interact with exposed TCPS surface can be explained by the formation of a BSA layer that does not cover the whole surface area and/or by exchange between already adsorbed BSA and HIS-tagged proteins in solution, where the HIS-tagged proteins have a higher affinity for the surface once adsorbed. Apart from the possibility that the HIS-tag itself leads to an increased binding strength, which we have generally considered in this paper, HIS-tagged proteins might also increase the ability of the protein to interact with exposed TCPS surface through an altered protein conformation. Some 3-D models of HIS-tagged proteins show that the HIS-tag is pointing out from the protein as an elongated structure [18]. An elongated structure like this might enhance the possibility for the HIS-tag to come in close contact with small areas of exposed TCPS surface between adsorbed BSA molecules compared to the proteins without a HIS-tag. This possibility further adds to the demand for consideration of BSA adsorption onto TCPS and PS. Based on the surface characteristics of PS and TCPS, one can expect the BSA molecules to undergo more relaxation on the rather hydrophobic PS surface compared to the more hydrophilic TCPS surface. The BSA proteins are therefore expected to obtain a larger contact area on and become more strongly associated with PS than with TCPS [19]. Thus, the BSA blocking might be less efficient on the TCPS surface than on the PS surface simply due to the fact that the BSA molecules have smaller contact area with the TCPS surface. This could also result in higher exchange ratio between BSA and HIS-tagged proteins on TCPS compared to PS. During the radioactive labelling experiments, not only adsorption onto PS and TCPS of the proteins under investigation, but also the adsorption of BSA, was detected (data not shown). The level of protein adsorption for BSA as well as for the protein of interest was detected on unblocked surfaces and compared to those surfaces subject to blocking. The results revealed comparative levels of BSA adsorption on either surface, independent on whether a prior BSA blocking was performed or not. This indicates that the presence of 1 mg/ml BSA as carrier protein in the solutions is sufficient to obtain monolayer coverage of BSA and that the increased adsorption of HIS-tagged proteins is not an artefact caused by an in-adequate BSA blocking procedure.
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Fig. 6. Adsorption of 1 g/ml SA-HIS onto TCPS when using no block or block with 1 mg/ml BSA, IgG or Fg. Blocking with BSA or IgG does not result in a significant reduction of SA-HIS adsorption onto TCPS, while blocking with Fg does. The ELISA signal is linearly correlated to amount protein adsorbed and the signal has been normalized. Surfaces were blocked for 30 min before adsorption, and 1 mg/ml BSA was used as carrier protein during adsorption of 1 g/ml SA-HIS (30 min). ELISA was performed according to the protocol described in Section 2.
Some proteins adsorb in a multilayer fashion on some, typically hydrophobic, polymer surfaces [20]. If this was the case with the BSA blocking, one would expect that some of the BSA present as carrier protein during adsorption of the PG and PG-HIS, would adsorb on top of the already adsorbed BSA layer on the blocked surfaces. Thus, surfaces subject to blocking should show higher levels of BSA adsorbed than the unblocked surfaces. This was not observed on PS or TCPS. This is again in correspondence with our hypothesis that the increased adsorption of HIS-tagged proteins is caused by electrostatic interaction, as physical contact between protein and surface does not seem likely if multilayer adsorption of BSA protein had been observed. These findings in total suggest that exchange between adsorbed BSA and HIS-tagged proteins in solution does in fact take place, and that the HIS-tagged proteins show a higher affinity for the surface once adsorbed, compared to the proteins without HIS-tag. The impact of blocking is also illustrated in Fig. 6, showing SAHIS adsorption onto TCPS after either no blocking or blocking with 1 mg/ml BSA, IgG or Fg for 30 min. SA-HIS was chosen here instead of PG-HIS to avoid interaction between PG-HIS and IgG molecules, which would be difficult to distinguish from unspecific adsorption of PG-HIS on the TCPS surfaces in the subsequently performed ELISA analysis. In Fig. 6 there is no significant difference between SA-HIS adsorption onto a TCPS surface that has not been blocked and onto a TCPS surface that has been blocked with BSA, illustrating that a pre-adsorption with BSA compared to only having BSA present as a carrier protein does not have a large impact on the level of adsorption of SA-HIS. Furthermore, blocking the surface with BSA or IgG seems to be rather ineffective in reducing the adsorption of SA-HIS onto TCPS. On the other hand, the use of fibrinogen as a blocking agent reduces the adsorption of SA-HIS onto TCPS significantly. Thus, our studies show that one could consider other proteins than BSA as blocking agents and that fibrinogen could be considered as an alternative blocking molecule, especially if the reduction of unspecific adsorption of proteins is the main objective. Fibrinogen is a rather large molecule (340 kDa) with an elongated shape and with structural characteristics that are expected to result in more flexibility in the arrangement of the molecules, when adsorbed onto the surface. Fibrinogen is also known to adsorb in higher concentrations onto TCPS than albumin [21]. Our studies show significantly increased adsorption of HIStagged PG, SA and VEGF, which all are proteins with pI below 7.4, onto TCPS. All investigations carried out support the
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hypothesis that the higher adsorption is primarily caused by electrostatic interactions between carboxylate groups on the TCPS surface and histidine residues in the HIS-tagged proteins. For IL4, which is a protein with pI above 7.4, a more complex adsorption pattern is observed and there is no conclusive tendency regarding adsorption of the HIS-tagged version of this protein onto TCPS and PS. It is possible that the smaller size of the IL4 protein, compared to the other proteins investigated, could result in a higher degree of flexibility with regard to finding exposed areas on the TCPS and that this effect is independent on the presence of a HIS-tag [22]. Another possibility is that the HIS-tag is inducing structural changes in the IL4 molecule that lowers adsorption of the HIS-tagged version of the protein compared to the native one. One could consider if the less pronounced increase in adsorption of VEGF after addition of the HIS-tag, compared to what is that observed for PG and SA, is influenced by the orientation of the protein relative to the TCPS surface. The existence of an overall negative charge of the PG-HIS and SA-HIS, combined with a small positive charge in one end of the molecule, could result in a situation where the negatively charged parts of the proteins are repelled by the negatively charged TCPS surface, thereby orienting the proteins with the HIS-tag facing the surface. The more neutral character of the VEGF protein cannot induce this preferred orientation of the HIS-tag pointing towards the surface and thereby not contribute to an increased possibility for the HIS-tag to come in physical contact with the TCPS surface and for electrostatic interactions to come into play. However, additional studies of other proteins with pI above 7.4 are needed to explore these aspects. Besides from illustrating how HIS-tagging may greatly influence adsorption properties of the tagged proteins with regard to adsorption to TCPS surfaces, the present study also illustrates that BSA blocking is not an efficient method for decreasing adsorption of HIS-tagged proteins onto these surfaces. Those findings are of importance in regards to experimental setups where a high degree of protein adsorption onto surfaces can cause changes in protein concentration in solution, for example biological assays where low protein concentrations are used. Thus, not only effects on biological function but also adsorption characteristics of proteins modified with a HIS-tag should be considered when using those in biological assays. 4. Conclusions Proteins with pI values below or around the pH at which adsorption is performed (pH 7.4) show higher adsorption onto TCPS when they are HIS-tagged than when they are adsorbed in the native form. The effect is not observed for the protein with a pI value above 7.4 or for any of the proteins when they are adsorbed onto PS. Results from ELISA and radioactive labelling, including adsorption studies at different pH values and at different ionic strengths, strongly indicate that the higher adsorption primarily is caused by electrostatic interaction between carboxylate groups in exposed areas of the TCPS surface and histidine residues in the proteins. These findings identify a possible problem when carrying out biological assays using protein solutions of low concentration, where a high adsorption of proteins to sample surfaces can change protein concentration in solution, and thus have an impact on the outcome of the assay. References [1] S.T Loughran, D. Walls, Purification of poly-histidine-tagged proteins, Methods Mol. Biol. 681 (2011) 311–335. [2] J. Caswell, P. Snoddy, D. McMeel, R.J Buick, C.J Scott, Production of recombinant proteins in Escherichia coli using an N-terminal tag derived from sortase, Protein Expr. Purif. 70 (2010) 143–150.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Increased adsorption of histidine-tagged proteins onto tissue culture polystyrene Maria Holmberg 1 , Thomas Steen Hansen 2 , Johan Ulrik Lind, Gertrud Malene Hjortø ∗ Department of Micro- and Nanotechnology, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark
a r t i c l e
i n f o
Article history: Received 13 October 2011 Received in revised form 2 December 2011 Accepted 2 December 2011 Available online 16 December 2011 Keywords: Histidine-tagged protein Adsorption Tissue culture polystyrene Electrostatic interaction Blocking molecule
a b s t r a c t In this study we compare histidine-tagged and native proteins with regards to adsorption properties. We observe significantly increased adsorption of proteins with an incorporated polyhistidine amino acid motif (HIS-tag) onto tissue culture polystyrene (TCPS) compared to similar proteins without a HIS-tag. The effect is not observed on polystyrene (PS). Adsorption experiments have been performed at physiological pH (7.4) and the effect was only observed for the investigated proteins that have pI values below or around 7.4. Competitive adsorption experiments with imidazole and ethylenediaminetetraacetic acid (EDTA), as well as adsorption performed at different pH and ionic strength indicates that the high adsorption is caused by electrostatic interaction between negatively charged carboxylate groups on the TCPS surface and positively charged histidine residues in the proteins. Pre-adsorption of bovine serum albumin (BSA) does not decrease the adsorption of HIS-tagged proteins onto TCPS. Our findings identify a potential problem in using HIS-tagged signalling molecule in assays with cells cultured on TCPS, since the concentration of the molecule in solution might be affected and this could critically influence the assay outcome. © 2011 Elsevier B.V. All rights reserved.
1. Introduction One of the most common procedures for purification of recombinant proteins is to incorporate a so-called histidine-tag (HIS-tag) in the protein amino acid sequence [1–3]. Such a HIS-tag typically consists of six histidine residues placed in either the N- or C-terminus of the protein. Histidine tagging enables efficient protein purification on an affinity column by chelation between the histidine residues and fixed transition metals such as Cu(II) and Ni(II) [4,5]. This method for purification of proteins is popular and many commercially available products have a polyhistidine amino acid motif incorporated into the protein. However, a number of reports have emerged regarding how the incorporation of a HIS-tag can result in unexpected side-effect such as the formation of protein hexamers [6], altered levels of catalytic activity in integrases [7], increased phosphorylation of proteins and subsequently resistance to thrombin cleavage [8], conformational changes [9] and assembly of proteins into sheet structures [10].
∗ Corresponding author. Tel.: +45 45 25 81 56. E-mail address: [email protected] (G.M. Hjortø). 1 Present address: Danish Technological Institute, Gregersensvej, Taastrup, Denmark. 2 Present address: Radiometer Medical ApS, Åkandevej, Brønshøj, Denmark. 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.12.001
In the present study we report another side-effect, namely that the incorporation of a HIS-tag in proteins can lead to significantly increased adsorption of the protein onto TCPS surfaces, compared to the native protein. TCPS is used in many different setups for cell culturing and is normally considered to be a standard cell compatible material [11,12]. An unexpected high adsorption of HIS-tagged proteins onto TCPS could potentially deprive the protein concentration in solution along with increasing the protein concentration at the surface. Such adsorption of proteins to the TCPS surface can thus have a large effect on the outcome of an assay, especially in biological assays where the effective concentrations of relatively small proteins, like growth factors and chemokines, most often lie in the range of a few ng/ml. The increased adsorption of HIS-tagged proteins was not seen on regular PS surfaces, indicating that hydrophobic interaction does not play a significant role in the observed phenomenon. In contrast to the rather hydrophobic and chemically inert PS surface, the TCPS surface contains a variety of functional groups, including carboxylate groups that are introduced to the surface by corona-discharge [13], making the surface hydrophilic. The increased adsorption of the HIS-tagged proteins onto the TCPS surface appears to be related to direct interactions between histidine residues in the HIS-tag and carboxyl acid groups on the TCPS surface, as adding imidazole or EDTA to the solutions results in decreased adsorption of HIS-tagged proteins. This direct interaction appears to be mainly of electrostatic character, between protonated histidine residues and de-protonated carboxylic acids. This was indicated by a notably
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decreased adsorption of HIS-tagged proteins when changing pH outside the physiologic range or when increasing the ionic strength of the buffer. The increased adsorption of HIS-tagged proteins onto TCPS was observed for proteins with a pI below 7.4. We suggest that for native proteins with pI below physiological pH, and thus with an overall negative surface charge at pH 7.4, the possibility for strong binding through electrostatic interaction between native protein and the negatively charged TCPS surface is low. The introduction of a HIS-tag in the protein makes a stronger interaction between the protein and the surface possible. In the case of a protein with pI above 7.4 and thus with an overall positive charge at physiological pH, even the native protein has the possibility for adsorbing to the TCPS surface through electrostatic interactions. For these proteins we do observe a slightly smaller adsorption of the HIS-tagged version, which might suggest that the overall positive surface charge at physiological pH either overshadow and/or disrupt interactions between HIS-tag and carboxyl acid groups on the TCPS surface. However, other factors such as protein conformation, protein size, and how the proteins interact with BSA carrier proteins can have an impact on adsorption characteristics of the proteins. In this study we have investigated the adsorption behaviour of a set of proteins, with and without HIS-tag, onto two different polymer surfaces, TCPS and PS. We show that adsorption of HIStagged proteins with a pI below 7.4 is much higher than adsorption of corresponding native proteins at pH 7.4 onto TCPS. We further investigate how adsorption is influenced by changing parameters such as pH, blocking procedure and ionic strength in buffer solution, using the independent techniques ELISA and radioactive labelling.
2. Materials and methods Proteins were purchased from R&D Systems (R&D Systems, USA), Abcam (Abcam, UK), Invitrogen (Invitrogen, USA), Sigma–Aldrich (Sigma–Aldrich, Germany) and BioVision (BioVision, USA) as presented in Table 1, also listing the theoretical values of molecular weight (MW) and isoelectric point (pI) of the proteins. Values for proteins used in both native and HIS-tagged form were obtained using the ‘ProtParam’ software available at www.expasy.com while values for BSA, Immunoglobulin G (IgG) and fibrinogen (Fg) are citations from literature [14,15]. Phosphate buffered saline (PBS) from Lonza (Lonza Copenhagen ApS, Denmark) with 1 mg/ml BSA (Sigma–Aldrich, Germany) was used for making protein solutions. IgG and Fg were derived from human plasma (Sigma–Aldrich, Germany). Goat anti-human IL4 antibody was from R&D Systems (cat. no. AF-204-NA), Rabbit anti-human VEGF antibody was from Abcam (cat. no. ab46154), Rabbit antiStreptavidin antibody was from Abcam (cat. no. ab59320). Rabbit anti-Goat IgG – HRP (H+L) was from Invitrogen (cat. no. 611620) and Goat anti-Rabbit IgG (H+L) was from KPL (cat. no. 474-1506). For ELISA experiments TCPS and PS 96 well ELISA plates from NUNC (Thermo Fisher Scientific, Denmark) was used. ELISA adsorption experiments were carried out using protein solutions with 1 mg/ml BSA as carrier protein. Experiments were performed in quadruplicates and at least two independent experiments were performed for each protein. The results presented are average values from one experiment with four replicates, where the error bars represents the standard deviation of the mean. TCPS and PS ELISA plate wells were incubated with BSA solution (200 l, 1 mg/ml) for 30 min. Subsequently the plates were washed three times in PBS buffer containing 0.1% Tween 20 and finally once with MilliQ water (this general washing procedure was used in all further washes). Protein adsorption, either with native or HIS-tagged protein, was carried out for 30 min (100 l, 1 g/ml unless otherwise stated). The surfaces were washed again
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as described above. Primary antibodies were added to the wells (100 l, 1 g/ml) and the reaction between adsorbed protein and antibody was allowed to proceed for 30 min before the surfaces were washed again. For ELISA with PG and PG-HIS, no primary antibody was used since the conjugated antibody can bind directly to Protein G molecules. The reaction with HRP-conjugated antibody (IgG-HRP) was carried out for 30 min (100 l, concentrations as recommended by suppliers). The plates were washed and allowed to react with 50 l TMB (3,3 ,5,5 -tetramethylbenzidine) (Sigma–Aldrich) for 3 min (if not otherwise stated). The enzymatic reaction was stopped by adding 0.2 M sulphuric acid. The colour development in each well was detected using an ELISA plate reader (VICTOR, Perkin Elmer, USA) measuring the absorbance at 450 nm for 1 s in each well (CW lamp filter P450). The TCPS and PS plates (96 well) used in radioactive labelling experiments were the same kind as those used in ELISA experiments. The Iodo-Gen method (Perkin-Elmer Life and Analytical Science, USA) was used to radiolabel the proteins with radioactive iodine, where 0.2 ml protein was mixed with 0.02 ml radioiodine and kept in the Iodo-Gen tube for 20 min before the solution was removed. PG and PG-HIS was labelled with 131 I and BSA was labelled with 125 I. The labelled proteins were separated from free iodine and other chemical reagents using gel chromatography (PD10 desalting column, Amersham Bioscience, UK). Concentrations of labelled proteins were calculated using the amount of added protein and volume of final solution, typically resulting in a concentration of a couple of hundreds of g/ml. Loss of protein during separation was approximated to be around 20%. The amount of free radioiodine in solutions with labelled protein was determined using TCA (trichloroacetic acid) precipitation, revealing the free radioiodine in solution to be around 5%. Adsorption onto blocked and unblocked surfaces was performed at 20 ◦ C and labelled BSA was used as carrier protein. For blocked surfaces, 200 l of 1 mg/ml BSA was added to the wells of a 96 well ELISA plate and allowed to adsorb for 30 min before washing (the same washing procedure as that used during ELISA experiments). 100 l of 1 g/ml PG or PG-HIS was added to the wells and allowed to adsorb for 30 min before the surfaces were washed again. Each substrate was placed in a clean plastic bag and radioactivity was detected using a gamma counter (Canberra Gamma Counter, USA). The radioactivity of 125 I and 131 I on each surface was measured by counting the 27–36 keV gamma rays of 125 I and the 364 keV gamma ray of 131 I. The contribution of 131 I isotope to the counting area of 125 I has been subtracted. The counting efficiency of the detector for 131 I (<5%) is lower than for 125 I (∼50%), and contribution of 131 I to the total 125 I counts in the 26–37 keV energy range is normally lower than 10%. By counting radioactivity of 125 I and 131 I in standard solutions (Ast ) with known amount of protein (mst ), the detected radioactivity on sample surface (As ) can be transformed to amount protein adsorbed (ms ) by using the formula; ms = (mst /Ast ) × As . Each adsorption experiment was performed in triplicates and the results presented are average values of measurements on three samples, with the error bars representing the standard deviation of the mean.
3. Results and discussion Fig. 1 displays results from ELISA analysis on adsorption of (a) PG, (b) SA, (c) VEGF and (d) IL4, all in a native and a HIS-tagged version, onto PS and TCPS. PG-HIS, SA-HIS and VEGF-HIS adsorb to TCPS to a significantly higher degree than the untagged versions of the same proteins. This effect is not seen in the case of the PS surface, where the proteins adsorb at a comparable and lower level, regardless of the presence or absence of HIS-tag in the proteins. In the case of IL4, the untagged protein adsorbs to a higher degree than the untagged
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Table 1 Abbreviations, supplier and theoretical values of proteins used in the study. The theoretical values of protein that were used in both native and HIS tagged form were evaluated using the ‘ProtParam’ software available at: www.expasy.org, using the amino acid sequence of the supplier. Theoretical values of BSA, IgG and Fg are based on references as indicated in the table. Protein
Interleukin-4 Interleukin-4 with a HIS-tag Vascular endothelial growth factor Vascular endothelial growth factor with a HIS-tag Streptavidin Streptavidin with a HIS-tag Protein G Protein G with a HIS-tag Bovine serum albumin Immunoglobulin G Fibrinogen
Abbreviation
IL4 IL4-HIS VEGF VEGF-HIS SA SA-HIS PG PG-HIS BSA IgG Fg
onto both surfaces, and both IL4 and IL4-HIS adsorb more to TCPS than to PS. The higher adsorption of HIS-tagged proteins onto TCPS compared to proteins without a HIS-tag was also investigated using radioactive labelled proteins, as shown in Fig. 2. PG and PG-HIS was labelled with radioactive iodine and adsorbed onto TCPS and PS using a similar protocol as that used during ELISA experiments (see Section 2). The radioactivity on the samples was detected and
Theoretical values
Supplier (cat. no.)
MW (Da)
pI
14963.2 15,786 19094.8 19917.6 65963.6 69254.8 21131.2 21,954 67,000 [15] 150,000 [15] 340,000 [15]
9.26 9.26 7.75 7.63 6.1 6.77 4.48 4.81 4.8 [14] 6.4–7.2 [14] 5.5 [14]
R&D (204-IL) Abcam (ab53342) R&D (293-VE) Abcam (ab55566) Invitrogen (S888) Abcam (78833) Sigma–Aldrich (194559) Biovision (6510-5) Sigma–Aldrich (85040C) Sigma–Aldrich (I4506) Sigma–Aldrich (F4883)
the corresponding amount of adsorbed protein on each surface was calculated. This experiment confirmed that HIS-tagged PG adsorbs to TCPS to a significantly higher degree than the untagged version of the same protein and that both proteins (PG and PG-HIS) show similar low adsorption onto PS. Data presented in Figs. 1 and 2, indicate that the HIS-tagged versions of PG and SA have a higher affinity for the negatively charged, hydrophilic TCPS surface, than for the PS surface. PG and SA have
Fig. 1. Adsorption of (a) PG and PG-HIS, (b) SA and SA-HIS, (c) VEGF and VEGF-HIS, and (d) IL4 and IL4-HIS onto TCPS and PS. PG-HIS, SA-HIS, and VEGF-HIS show higher adsorption than PG, SA and VEGF onto TCPS, while the effect is not observed on PS. In the case of IL4, the protein without a HIS-tag shows higher adsorption than the protein with a HIS-tag on both TCPS and PS. The presented data are normalized signals from the VICTOR ELISA plate reader (Perkin Elmer, USA) and there is a linear correlation between the signal and the amount of adsorbed protein. Surfaces were blocked with 1 mg/ml BSA for 30 min before adsorption, and 1 mg/ml BSA was used as carrier protein during adsorption of 1 g/ml of investigated protein (native and HIS-tagged) (30 min). Afterwards the samples were washed and ELISA was performed according to the protocol described in Section 2.
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Fig. 2. Adsorption of PG and PG-HIS onto TCPS and PS detected using radioactive labelling, showing a significantly higher adsorption of PG-HIS than PG onto TCPS while both proteins show similar and lower level of adsorption onto PS. Surfaces were blocked with 1 mg/ml BSA for 30 min before adsorption, and 1 mg/ml BSA was used as carrier protein during adsorption of 1 g/ml PG and PG-HIS (30 min).
isoelectric points below 7.4 (see Table 1), the pH at which the experiments are carried out, and thus have an overall negative charge during adsorption. We suggest that when introducing a HIS-tag in the proteins, a local concentration of positive charges that can take part in electrostatic interactions with the carboxylate groups on the TCPS surface is added to the proteins, and that this is the principle cause of the increased adsorption, seen for these HIS-tagged proteins. VEGF shows a similar adsorption pattern as PG and SA, with higher adsorption of VEGF-HIS than VEGF on TCPS and the same level of adsorption of the two proteins on the PS surface. However, the difference in adsorption between VEGF and VEGF-HIS onto TCPS is smaller compared to what is observed for PG and SA. VEGF has a theoretical isoelectric point just above 7.4 (see Table 1) and can be expected to be overall neutral, with tendency to have a small positive charge, at physiological pH. This might explain the higher adsorption of the native protein and the less pronounced effect of the addition of a HIS-tag to VEGF regarding the adsorption onto TCPS. For IL4, with a pI well above 7.4 and an overall positive charge at the experimental conditions used, adding a HIS-tag to the protein does not necessarily improve the interaction with the TCPS surface. Both IL4 and IL4-HIS can thus be expected to show a rather high adsorption onto TCPS caused by electrostatic interaction between protein and surface.
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To investigate if the increased binding of HIS-tagged proteins onto TCPS was caused by direct interaction between the HIS-tag and the TCPS substrate, experiments with competitive adsorption were performed. Fig. 3 presents the adsorption of 10 ng/ml PG-HIS onto TCPS in competition with (a) imidazole and (b) EDTA. Imidazole is an organic compound with molecular weight of approximately 68 Da,which is identical to the ring structure present in the histidine side chain. EDTA is a poly-acid containing four carboxylate groups, and a strong chelator of divalent cations. The latter should be a negligible effect in our studies as buffers without Mg2+ and Ca2+ were generally used. From the above, one can expect that imidazole can compete with the HIS-tag in the modified proteins for interacting with the TCPS surface, while EDTA can compete with the carboxylate groups on the TCPS surface for interacting with the HIS-tag. As observed in Fig. 3, adsorption of PG-HIS onto TCPS decreases with increased concentration of both imidazole and EDTA. All EDTA concentrations tested resulted in a significant reduction of protein adsorption. At EDTA concentrations below 10 mM, the reduction in protein adsorption is probably due to competition between the EDTA groups and the carboxylates on the surface for interaction with the HIS-tag on the proteins. A further drop, as that observed for 100 mM EDTA could also be partly an effect of the increased ionic strength of the solution achieved by the high concentration of EDTA molecules present at this concentration. The effect of adding higher concentrations of imidazole strongly suggest that the histidine residues in the proteins are involved in the higher adsorption of the HIS-tagged proteins. The suggestion that the higher adsorption is caused by electrostatic interaction between histidine residues and carboxylate groups was further investigated looking at adsorption of HIS-tagged proteins as a function of pH and ionic strength in the buffer. A change in pH will result in changes in the level of protonation of carboxylate groups on the TCPS surface and of the histidine residues in the protein, while an increase in the ionic strength will shield electrostatic interactions in general. In Fig. 4, adsorption of 100 ng/ml PG and PG-HIS onto TCPS is shown as a function of pH (pH 4–9). Reaction with TMB during ELISA experiments was carried out approximately 6½ times longer for PG than for PG-HIS. The longer reaction time for PG was necessary to obtain a strong enough absorbance signal for ELISA reader detection. The weak signal was due to the much lower adsorption of native PG compared to PG-HIS. As observed in Fig. 4, an overall higher adsorption of PG-HIS compared to PG onto TCPS was observed at all pH values. Furthermore, there was a shift of maximum adsorption to a higher pH value for PG-HIS compared to PG. The carboxylate groups on the TCPS
Fig. 3. Adsorption of PG-HIS onto TCPS in the presence of different concentrations of (a) imidazole and (b) EDTA showing the normalized signal from the ELISA plate reader for which there is a linear correlation between the signal and the amount of adsorbed protein. The adsorption of PG-HIS decreases with increased concentration of both imidazole and EDTA, indicating that the high adsorption of PG-HIS relies on interaction between the histidine residues in the protein and the carboxylate groups on the TCPS surface. Surfaces were blocked with 1 mg/ml BSA for 30 min before solutions of 10 ng/ml HIS-tagged protein, carrier protein (1 mg/ml BSA) and either imidazole or EDTA were added to the surface (30 min). Afterwards the samples were washed and ELISA was performed according to the protocol described in Section 2.
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Fig. 4. Adsorption of 100 ng/ml PG and PG-HIS onto TCPS as a function of pH. The adsorption of PG-HIS is generally higher than the adsorption of PG and the maximum adsorption for PG-HIS is positioned at a higher pH than the maximum value of PG. The signal from the ELISA reader, which is linearly correlated with the amount protein adsorbed, has been normalized. It should be noted that for obtaining a detectable signal from PG adsorption, the TMB reaction step had to be carried out for a time period that was more than 6 times longer than that for PG-HIS adsorption. Otherwise the adsorption protocol for PG and PG-HIS was identical and 30 min of 1 mg/ml BSA blocking was performed before 30 min of adsorption of PG or PG-HIS with BSA (1 mg/ml) as carrier protein was carried out.
surface can be expected to have a pKa around 5 and HuyghuesDespointes et al. [16] report that the pKa of histidine is 6.5–7.0 when forming ionic pairs with a carboxylic acid (aspartate). Thus, at pH below 5 the carboxylic acids are increasingly protonated and at pH above 7.0 the histidine residues are de-protonated. This correlates well with the drop in adsorption of PG-HIS onto TCPS below pH 5 and above pH 7.4 strengthening the hypothesis that the high adsorption is caused by electrostatic interaction between carboxylate groups on the TCPS surface and histidine residues in the protein. The shift of maximum adsorption to a higher pH value for PG-HIS can be expected by a shift to a higher pI for the HIS-tagged protein compared to the protein without a HIS-tag (see Table 1). The electrostatic attraction between the carboxylic acid and HIStag is expected to be inversely proportional to the ionic strength of the solution due to increased shielding [17]. Experiments were conducted with varying ionic strength to verify that electrostatic attraction is the main mechanism in the observed adsorption of histidine-tagged proteins. Fig. 5 shows adsorption of 100 ng/ml PG-HIS onto TCPS as a function of NaCl concentration (0–800 mM) in the buffer. Here, a dramatic decrease in adsorption is observed when the NaCl concentration increases from 0 mM to 800 mM. The experiments with pH and ionic strength support that the main mechanism responsible for the increased adsorption of HIS-tagged proteins is electrostatic attraction. BSA blocking is a universal method for decreasing unspecific adsorption of proteins to surfaces. The experimental conditions used in this study are similar to conditions used within ELISA, as well as many other biological assays where BSA blocking is employed. Thus it is relevant to consider the influence of BSA blocking on the observed adsorption patterns. In all experiments performed, BSA has been used as a carrier protein and is thus present in a much higher concentration (mg/ml) than the proteins investigated for unspecific adsorption onto PS and TCPS (ng-g/ml). Furthermore, in all ELISA experiments presented surfaces have been blocked with 1 mg/ml BSA for 30 min before performing adsorption with the proteins that are under investigation. As electrostatic interactions are short range in aqueous environments, the proteins need to be in physical contact with the surface for these interactions to come into play, and establish a binding
Fig. 5. Adsorption of PG-HIS onto TCPS as a function of NaCl concentration in the buffer showing a decrease in adsorption with increased ionic strength. The signal from the ELISA reader correlates linearly with amount protein adsorbed on the surface and has been normalized. The surface has been blocked with 1 mg/ml BSA for 30 min before adsorption with 100 ng/ml PG-HIS in PBS buffer (with 1 mg/ml BSA as carrier proteins) with different NaCl concentrations was carried out.
that withstands the rinsing procedures used in this study. The high level of HIS-tagged proteins on TCPS thus indicates that there has to be exposed areas of the polymer surface during the incubation. The possibility for HIS-tagged proteins to interact with exposed TCPS surface can be explained by the formation of a BSA layer that does not cover the whole surface area and/or by exchange between already adsorbed BSA and HIS-tagged proteins in solution, where the HIS-tagged proteins have a higher affinity for the surface once adsorbed. Apart from the possibility that the HIS-tag itself leads to an increased binding strength, which we have generally considered in this paper, HIS-tagged proteins might also increase the ability of the protein to interact with exposed TCPS surface through an altered protein conformation. Some 3-D models of HIS-tagged proteins show that the HIS-tag is pointing out from the protein as an elongated structure [18]. An elongated structure like this might enhance the possibility for the HIS-tag to come in close contact with small areas of exposed TCPS surface between adsorbed BSA molecules compared to the proteins without a HIS-tag. This possibility further adds to the demand for consideration of BSA adsorption onto TCPS and PS. Based on the surface characteristics of PS and TCPS, one can expect the BSA molecules to undergo more relaxation on the rather hydrophobic PS surface compared to the more hydrophilic TCPS surface. The BSA proteins are therefore expected to obtain a larger contact area on and become more strongly associated with PS than with TCPS [19]. Thus, the BSA blocking might be less efficient on the TCPS surface than on the PS surface simply due to the fact that the BSA molecules have smaller contact area with the TCPS surface. This could also result in higher exchange ratio between BSA and HIS-tagged proteins on TCPS compared to PS. During the radioactive labelling experiments, not only adsorption onto PS and TCPS of the proteins under investigation, but also the adsorption of BSA, was detected (data not shown). The level of protein adsorption for BSA as well as for the protein of interest was detected on unblocked surfaces and compared to those surfaces subject to blocking. The results revealed comparative levels of BSA adsorption on either surface, independent on whether a prior BSA blocking was performed or not. This indicates that the presence of 1 mg/ml BSA as carrier protein in the solutions is sufficient to obtain monolayer coverage of BSA and that the increased adsorption of HIS-tagged proteins is not an artefact caused by an in-adequate BSA blocking procedure.
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Fig. 6. Adsorption of 1 g/ml SA-HIS onto TCPS when using no block or block with 1 mg/ml BSA, IgG or Fg. Blocking with BSA or IgG does not result in a significant reduction of SA-HIS adsorption onto TCPS, while blocking with Fg does. The ELISA signal is linearly correlated to amount protein adsorbed and the signal has been normalized. Surfaces were blocked for 30 min before adsorption, and 1 mg/ml BSA was used as carrier protein during adsorption of 1 g/ml SA-HIS (30 min). ELISA was performed according to the protocol described in Section 2.
Some proteins adsorb in a multilayer fashion on some, typically hydrophobic, polymer surfaces [20]. If this was the case with the BSA blocking, one would expect that some of the BSA present as carrier protein during adsorption of the PG and PG-HIS, would adsorb on top of the already adsorbed BSA layer on the blocked surfaces. Thus, surfaces subject to blocking should show higher levels of BSA adsorbed than the unblocked surfaces. This was not observed on PS or TCPS. This is again in correspondence with our hypothesis that the increased adsorption of HIS-tagged proteins is caused by electrostatic interaction, as physical contact between protein and surface does not seem likely if multilayer adsorption of BSA protein had been observed. These findings in total suggest that exchange between adsorbed BSA and HIS-tagged proteins in solution does in fact take place, and that the HIS-tagged proteins show a higher affinity for the surface once adsorbed, compared to the proteins without HIS-tag. The impact of blocking is also illustrated in Fig. 6, showing SAHIS adsorption onto TCPS after either no blocking or blocking with 1 mg/ml BSA, IgG or Fg for 30 min. SA-HIS was chosen here instead of PG-HIS to avoid interaction between PG-HIS and IgG molecules, which would be difficult to distinguish from unspecific adsorption of PG-HIS on the TCPS surfaces in the subsequently performed ELISA analysis. In Fig. 6 there is no significant difference between SA-HIS adsorption onto a TCPS surface that has not been blocked and onto a TCPS surface that has been blocked with BSA, illustrating that a pre-adsorption with BSA compared to only having BSA present as a carrier protein does not have a large impact on the level of adsorption of SA-HIS. Furthermore, blocking the surface with BSA or IgG seems to be rather ineffective in reducing the adsorption of SA-HIS onto TCPS. On the other hand, the use of fibrinogen as a blocking agent reduces the adsorption of SA-HIS onto TCPS significantly. Thus, our studies show that one could consider other proteins than BSA as blocking agents and that fibrinogen could be considered as an alternative blocking molecule, especially if the reduction of unspecific adsorption of proteins is the main objective. Fibrinogen is a rather large molecule (340 kDa) with an elongated shape and with structural characteristics that are expected to result in more flexibility in the arrangement of the molecules, when adsorbed onto the surface. Fibrinogen is also known to adsorb in higher concentrations onto TCPS than albumin [21]. Our studies show significantly increased adsorption of HIStagged PG, SA and VEGF, which all are proteins with pI below 7.4, onto TCPS. All investigations carried out support the
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hypothesis that the higher adsorption is primarily caused by electrostatic interactions between carboxylate groups on the TCPS surface and histidine residues in the HIS-tagged proteins. For IL4, which is a protein with pI above 7.4, a more complex adsorption pattern is observed and there is no conclusive tendency regarding adsorption of the HIS-tagged version of this protein onto TCPS and PS. It is possible that the smaller size of the IL4 protein, compared to the other proteins investigated, could result in a higher degree of flexibility with regard to finding exposed areas on the TCPS and that this effect is independent on the presence of a HIS-tag [22]. Another possibility is that the HIS-tag is inducing structural changes in the IL4 molecule that lowers adsorption of the HIS-tagged version of the protein compared to the native one. One could consider if the less pronounced increase in adsorption of VEGF after addition of the HIS-tag, compared to what is that observed for PG and SA, is influenced by the orientation of the protein relative to the TCPS surface. The existence of an overall negative charge of the PG-HIS and SA-HIS, combined with a small positive charge in one end of the molecule, could result in a situation where the negatively charged parts of the proteins are repelled by the negatively charged TCPS surface, thereby orienting the proteins with the HIS-tag facing the surface. The more neutral character of the VEGF protein cannot induce this preferred orientation of the HIS-tag pointing towards the surface and thereby not contribute to an increased possibility for the HIS-tag to come in physical contact with the TCPS surface and for electrostatic interactions to come into play. However, additional studies of other proteins with pI above 7.4 are needed to explore these aspects. Besides from illustrating how HIS-tagging may greatly influence adsorption properties of the tagged proteins with regard to adsorption to TCPS surfaces, the present study also illustrates that BSA blocking is not an efficient method for decreasing adsorption of HIS-tagged proteins onto these surfaces. Those findings are of importance in regards to experimental setups where a high degree of protein adsorption onto surfaces can cause changes in protein concentration in solution, for example biological assays where low protein concentrations are used. Thus, not only effects on biological function but also adsorption characteristics of proteins modified with a HIS-tag should be considered when using those in biological assays. 4. Conclusions Proteins with pI values below or around the pH at which adsorption is performed (pH 7.4) show higher adsorption onto TCPS when they are HIS-tagged than when they are adsorbed in the native form. The effect is not observed for the protein with a pI value above 7.4 or for any of the proteins when they are adsorbed onto PS. Results from ELISA and radioactive labelling, including adsorption studies at different pH values and at different ionic strengths, strongly indicate that the higher adsorption primarily is caused by electrostatic interaction between carboxylate groups in exposed areas of the TCPS surface and histidine residues in the proteins. These findings identify a possible problem when carrying out biological assays using protein solutions of low concentration, where a high adsorption of proteins to sample surfaces can change protein concentration in solution, and thus have an impact on the outcome of the assay. References [1] S.T Loughran, D. Walls, Purification of poly-histidine-tagged proteins, Methods Mol. Biol. 681 (2011) 311–335. [2] J. Caswell, P. Snoddy, D. McMeel, R.J Buick, C.J Scott, Production of recombinant proteins in Escherichia coli using an N-terminal tag derived from sortase, Protein Expr. Purif. 70 (2010) 143–150.
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