Incorporation of short, charged peptide tags affects the temperature responsiveness of positively-charged elastin-like polypeptides

Abstract

Elastin-like polypeptides (ELPs) are recombinant protein domains exhibiting lower critical solution temperature (LCST) behavior. This LCST behavior is controlled not only by intrinsic factors including amino acid composition and polypeptide chain length but also by non-ELP fusion domains. Here, we report that the presence of a composite non-ELP sequence that includes both His and T7 tags or a short Ser-Lys-Gly-Pro-Gly (SKGPG) sequence can dramatically change the LCST behavior of a positively-charged ELP domain. Both the His and T7 tags have been widely used in recombinant protein design to enable affinity chromatography and serve as epitopes for protein detection. The SKGPG sequence has been used to improve the expression of ELPs. Both the composite tag and the SKGPG sequence are <15% of the total length of the ELP fusion proteins. Despite the small size of the composite tag, its incorporation imparted pH-sensitive LCST behavior to the positively-charged ELP fusion protein. This pH sensitivity was not observed with the incorporation of the SKGPG sequence. The pH sensitivity results from both electrostatic and hydrophobic interactions between the composite tag and the positively-charged ELP domain. The hydrophobicity of the composite tag also alters the ELP interaction with Hofmeister salts by changing the overall hydrophobicity of the fusion protein. Our results suggest that incorporation of short tag sequences should be considered when designing temperature-responsive ELPs and provide insights into utilizing both electrostatic and hydrophobic interactions to design temperature-responsive recombinant proteins as well as synthetic polymers.

Figure 1.

Production of the I-tag and S-tag[YKV] proteins. (A) Illustration of the differences between the I-tag and S-tag[YKV] proteins. All proteins shared a common ELP YKV domain. I-tag included the sequence of SKGPG. The S-tag was composed of a T7 tag, a 7x His tag, and an enterokinase cleavage site. (B) SDS-PAGE and Western blot images of purified I-tag and S-tag[YKV] proteins. All proteins on the SDS-PAGE gel were close to their expected molecular weights (I-tag[YKV-48]: 21 kDa, S-tag[YKV-48]: 25 kDa, S-tag[YKV-72]: 28.5 kDa, and S-tag[YKV-96]: 32 kDa). Western blot confirmed the presence of the T7 tag on the S-tag[YKV] proteins. Western blot was not applicable for I-tag[YKV-48] due to a lack of the T7 tag epitope used for detection.

Figure 2.

The T_t values of I-tag[YKV-48] were insensitive to pH, whereas the T_t values of S-tag[YKV-48] decreased drastically with increasing pH. T_t measurements of (A) I-tag[YKV-48] and (B) S-tag[YKV-48] at protein concentrations ranging from 50 to 400 μM and pH values ranging from 5.5 to 8.0. Both proteins had lower T_t values with increasing protein concentration. At a given concentration, the T_t values of I-tag[YKV-48] were similar at all pH values. On the other hand, S-tag[YKV-48] showed pH-sensitive T_t values. NaCl was added to the solution for all groups at a final concentration of 0.2 M. The T_t values were presented as the average with standard deviation of three independent samples.

Figure 3.

T_t values of I-tag[YKV-48] and S-tag[YKV-48] were well described by a two-parameter equation. T_t measurements with best-fit lines from equation 1 of (A) I-tag[YKV-48] and (B) S-tag[YKV-48]. The T_t values were presented as the average with standard deviation of three independent samples.

Figure 4.

Concentration dependence (b_pH) of S-tag[YKV-48] was pH sensitive. Fitted b_pH of I-tag and S-tag[YKV-48] with standard error were plotted as a function of pH. The b_pH values for I-tag[YKV-48] yielded a nearly horizontal line, and b_pH was not sensitive to pH. On the other hand, the b_pH values for S-tag[YKV-48] were sensitive to pH and exhibited two different trends below and above pH 7. Larger changes in the b_pH values were observed from pH 5.5 to 7.0, whereas smaller changes were seen from pH 7.0 to 8.0.

Figure 5.

Longer YKV domain lengths decreased the extent of pH sensitivity derived from the S-tag sequence. At 200 μM protein and 0.2 M NaCl, the change in pH sensitivity due to the extension of the YKV domain was more prominent at a lower pH region (pH 5.5 to 6.5). The data set of S-tag[YKV-48] is replotted from Fig. 2B. The T_t values were presented as the average with standard deviation of three independent samples.

Figure 6.

High ionic strength shielded the electrostatic interactions between S-tag and the YKV-48 domain. T_t measurements of (A) I-tag and (B) S-tag[YKV-48] at different NaCl concentrations with 200 μM protein. The LCST behavior of I-tag[YKV-48] remained insensitive to pH over all NaCl concentrations. On the other hand, the LCST behavior of S-tag[YKV-48] showed decreasing pH sensitivity with increasing NaCl concentration. In addition, the two proteins responded differently to the increasing NaCl concentration. The T_t values of I-tag[YKV-48] initially increased until 0.3 M NaCl then decreased. In contrast, the T_t values of S-tag[YKV-48] decreased monotonically with increasing NaCl concentration. T_t values were presented as the average with standard deviation of three independent samples.

Figure 7.

High protein concentration and high ionic strength decreased the T_t values of S-tag[YKV-48] more significantly than those of I-tag[YKV-48]. LCST differences between S-tag and I-tag[YKV-48] with different (A) protein concentrations and (B) NaCl concentrations. S-tag[YKV-48] had higher T_t values than I-tag[YKV-48] at low pH values and low protein concentrations. As the protein concentration increased, S-tag[YKV-48] only had a higher T_t at pH 5.5. With varying NaCl concentration, S-tag[YKV-48] only had higher T_t values at NaCl concentrations ≤0.2 M. The plotted differences were based on the averaged T_t values reported in Fig. 2 and 6.

Figure 8.

Non-ELP tag identity affected the interactions between ELPs and Hofmeister ions. T_t measurements of I-tag[YKV-48] and S-tag[YKV-48] with (A) Na₂SO₄ and (B) NaI. Both I-tag[YKV-48] and S-tag[YKV-48] had decreasing T_t values at >0.2 M Na₂SO₄. I-tag[YKV-48] showed an increasing trend in T_t from 0.1 to 0.2 M Na₂SO₄ even with a strong kosmotropic ion such as SO₄^2−. With NaI, both proteins exhibited a similar trend in which T_t values decreased <0.2 M NaI and increased at higher concentrations. T_t values with Na₂SO₄ were determined at a protein concentration of 50 μM at pH 5.5. T_t values with NaI were determined at a protein concentration of 200 μM at pH 7.5. The presented T_t values were averages of three independent samples with standard deviation.