Results

Experimental outcomes and protein characterization

Introduction

The project aimed to obtain and characterize four constructs corresponding to Protein A and Protein B, each in their normal and extended forms, with the extended versions including the cryptic peptide region (35 additional amino acids). Thus, the constructs are defined as follows:

  • Protein A normal form → referred to as 1 (75 kDa, the construct form 100 kDa)
  • Protein A extended form → referred to as 2 (construct form 100 kDa)
  • Protein B normal form → referred to as 3 (74 kDa, the construct form 100 kDa)
  • Protein B extended form → referred to as 4 (construct form 100 kDa)

Cloning

The cloning phase was successful (see Figure 1). Recombination and transformation processes were verified through electrophoresis (load 400 ng). We used for further experiments 1.2 / 2.5 / 3.3 / 4.3.

0.5% agarose gel electrophoresis of vector and constructs
Figure 1: 0.5% agarose gel electrophoresis of vector (V-) and constructs (vector + insert) to evaluate which minipreps from colony picking were positive (contain the insert) and are therefore suitable for sequencing.

Sequencing results were also confirmed that the recombination process was favorable. The gene corresponding to protein 3 contains two missense mutations: the first one in the GST tag, changing Isoleucine (Ile) to Methionine (Met), the second in the protein of interest, changing Aspartic acid (Asp) to Glycine (Gly). According to computational evaluation, both changes result in a minor energy alteration, therefore are unlikely to affect experimental outcomes.

Protein Expression

Protein expression was carried out in both BL21 and RIPL E. coli strains. Expression was analyzed for all four constructs by examining To (at 18 °C, prior to induction) and Tf (after overnight induction at 18 °C). Additionally, the presence of any truncated forms was evaluated.

Western blot analysis in a 4-12% gel
Figure 2: Western blot analysis in a 4-12% gel. A) Western blot for GST antibody using BL21, template: B) Western blot for GST antibody using RIPL, template: DC / P1 To / P1 Tf / P2 To / P2 Tf / P3 To / P3 Tf / P4 To / P4 Tf.

Constructs show detectable protein bands at the expected molecular weights in both Coomassie and Western blots, despite the presence of bands corresponding to truncated proteins or GST-tag alone. Protein 1 has fewer truncated proteins compared to proteins 3 and 4. As protein 1 and 2 are very similar, we...

Western blot analysis in a 4-12% gel for GST antibody
Figure 3: Western blot analysis in a 4-12% gel for GST antibody.

Bands were detected after being at 37 ºC and before passing bacteria to 18 ºC, at To (pre-induction, 18 ºC for 1h) and Tf (post-induction, 18 ºC O/N) samples, suggesting either potential toxicity of the protein to the bacteria or basal expression driven by the plasmid. This is further supported by Western blot images, where the To signal is stronger than Tf, contrary to expectations. However, one Western blot (Figure 3) showed optimal protein expression across the samples, suggesting that the proteins are not toxic to the bacteria. No further conclusions can be drawn from this result.

Protein Purification

We purified the protein of interest using both 6HIS and GST affinity methods.

His-tag proteins were detectable in the elution fractions. Coomassie staining revealed significant protein contamination, as expected, since many endogenous proteins contain His-rich regions. Single-step His-tag purification proved insufficient to achieve the purity required.

Coomassie-stained SDS-PAGE analysis on a 4-12% gel
Figure 4: Coomassie-stained SDS-PAGE analysis on a 4-12% gel. Fractions were collected during affinity column purification, with fraction 3 (F3) showing the highest protein abundance.

GST-tag purification shows less protein contamination compared to His-tag method. However, we are not able to have the protein of interest isolated completely, as truncated proteins, mainly GST, are present. These truncations are more pronounced in constructs 3 and 4.

Coomassie-stained SDS-PAGE analysis on a 4-12% gel
Figure 5: Coomassie-stained SDS-PAGE analysis on a 4-12% gel. Analyses of the protein purifications.

Purified protein quantification revealed a low 260/280 ratio, pointing to the presence of impurities or incomplete removal of nucleic acids. The integrity of the constructs 3 and 4 was confirmed through plasmid digests and sequencing, ruling out degradation of the plasmid as the cause of truncations. Thus, the instability appears to originate at the protein level.

Despite the challenges, our results demonstrate that both protein constructs can be expressed and purified. Protein B constructs show a stronger tendency toward truncation.

The combination of His and GST purification strategies in future assays may help improve protein purity.