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Production of recombinant TEV protease in Escherichia coli and analysis of enzymatic reactions

Nerjovaj, Fisnik LU (2024) KBTM05 20241
Biotechnology
Biotechnology (MSc)
Abstract
Although different types of affinity tags have become a tool in the production and purification of recombinant proteins in various applications, the use of these have the risk of interfering with the biological activity and crystallization of proteins.There are both chemical and enzymatic ways to remove these tags, the enzymes include enteropeptidase, thrombokinase and protease to mention a few. The use of affinity tag-removing proteases has specifically proven to be useful. One of these enzymes include Tobacco Etch Virus (TEV) protease which recognizes the amino acids sequence ENLYFQ*G/S and cleaves between the Q and G/S, making it ideal for precise protein processing. Some limitations to these enzymes, like most other, are the fact that... (More)
Although different types of affinity tags have become a tool in the production and purification of recombinant proteins in various applications, the use of these have the risk of interfering with the biological activity and crystallization of proteins.There are both chemical and enzymatic ways to remove these tags, the enzymes include enteropeptidase, thrombokinase and protease to mention a few. The use of affinity tag-removing proteases has specifically proven to be useful. One of these enzymes include Tobacco Etch Virus (TEV) protease which recognizes the amino acids sequence ENLYFQ*G/S and cleaves between the Q and G/S, making it ideal for precise protein processing. Some limitations to these enzymes, like most other, are the fact that it has auto-cleaving properties which reduces the enzyme stability. In addition, the enzyme has solubility problems when it is produced by Escherichia coli. This can be circumvented by creating a fusion form of the protease with the chaperone MBP (maltose-binding-protein) which also prevents the auto-cleaving function. This thesis describes the production of a recombinant form of the Tobacco Etch Virus protease, analysis of the enzymatic reactions this enzyme has with different substrates and looks at various applications the obtained peptides can have. The substrates include a synthetic polypeptide created with the characteristic recognition site of TEV protease. The other substrate was β-casein which has a similar looking amino acid sequence compared to the recognition site for TEV protease. The methods leading this investigation included computational methods such as the visualisation and molecular modelling tool UCSF Chimera, where a docking could be achieved showing the interaction between the TEV protease and the substrates. Another computational tool included the use of A Plasmid Editor (ApE) program to construct the recombinant genes used for successful cultivation of the host organism E. coli and the expression genes for the produced polypeptides. The practical methods included a colorimetric assay with Folin-Ciocalteu’s reagent showing activity based on a reduction reaction led by reducing phenolic compounds in β-casein, SDS-PAGE and mass spectrometry analysis of the hydrolysed polypeptides. All results showed that TEV protease had activity and was not as specific as previously thought since a hydrolysis of the β-casein could be observed. The hydrolysis of the synthetic polypeptide could play a beneficial role in the future when manufacturing therapeutic agents since the peptides derived from the synthetic polypeptide after digestion have dual-inhibiting bioactivity against hypertension and type-2 diabetes. (Less)
Popular Abstract
Recent experiments, investigated in this thesis, may have shown that the TEV protease sequence recognition specificity is not as stringent as previously thought. This due to the possible observed hydrolysis of the substrate β-casein when digested with recombinant TEV protease. This new discovery may lead to new opportunities when using TEV protease as a tool in the lab as well as unexpected challenges. The stringent sequence specificity of TEV protease has been studied by many and it is well-known by now that this recognition sequence consists of the amino acids ENLYFQ*G/S (where the asterisk marks the point of hydrolysation). The G, at the P1’ position, can be substituted by several other amino acids, including S (Serine), without... (More)
Recent experiments, investigated in this thesis, may have shown that the TEV protease sequence recognition specificity is not as stringent as previously thought. This due to the possible observed hydrolysis of the substrate β-casein when digested with recombinant TEV protease. This new discovery may lead to new opportunities when using TEV protease as a tool in the lab as well as unexpected challenges. The stringent sequence specificity of TEV protease has been studied by many and it is well-known by now that this recognition sequence consists of the amino acids ENLYFQ*G/S (where the asterisk marks the point of hydrolysation). The G, at the P1’ position, can be substituted by several other amino acids, including S (Serine), without compromising the recognition pattern for the enzyme. The TEV protease has an auto-lysis tendency which is important to counter and can be done by fusing the fusion protein MBP (maltose-binding protein) to the protease, which prevents this detrimental function. The typical activity for the enzyme is to hydrolyse peptide bonds between Q (Glutamine) and G (Glycine), making the protease usable as a tool for precise removal of affinity tags. Recent experiments however have possibly shown that this specificity is not as strict as previously considered. The investigation in this thesis was conducted on the protein β-casein from bovine milk (UniProt accession no. P02666), which has an amino acid sequence similar to the recognition site for TEV protease between amino acids 131-142 (ENLHLPLPLLQS for β-casein compared to ENLYFQG for classic TEV protease recognition). The investigation started by docking the 12 amino acid long ligand to TEV protease which acts as the receptor in the UCSF Chimera program, which is a software allowing in-depth visualisation of molecular structures. The results showed a possible interaction between the thiol part of the cysteine (Cys151) in the active site of TEV protease to the carbonyl atom between amino acid residues Q and S on the β-casein ligand. This discovery led to the need for more experimental assays in the lab to further strengthen the results. After successfully producing the recombinant his-tagged TEV protease in the host organism E. coli, the first assay to confirm a digestion of β-casein was the colorimetric assay using Folin-Ciocalteu´s phenol reagent. The reagent consists of the two acids phosphomolybdate and phosphotungstate, which are yellow in their oxidized form but turn blue after reacting with reducing phenolic compounds. The phenolic compounds are usually released upon digestion of substrates by enzymes. The blue colour is directly proportional to the concentration of the released reducing phenolic compounds and can be measured using a spectrophotometer to measure absorbance. The quantification can be done by comparing to a phenolic compound used as a standard with known concentrations. In this case, tyrosine was used as a standard to measure the proteolytic activity. The assay showed that a phenolic compound was released upon interaction between β-casein and TEV protease, however further investigation was needed to confirm a digestion by the enzyme. Another assay used was SDS-PAGE technology, which showed slightly more bands on the β-casein control (undigested protein) compared to digested β-casein. To see if these bands corresponded to a peptide obtained after cleavage with TEV protease, a more detailed evaluation had to be done using MALDI-TOF mass spectrometry. At first glance, the results from these showed very little difference between the β-casein control (undigested) and the digested β-casein, however a peak at approximately 17.5 kDa could be observed on the digested sample of β-casein and not (or at least much less) on the unhydrolyzed control. Another small peak was observed at 7649.959 Da in the digested sample. Both polypeptide fragments obtained after digestion correspond well to the weights (when checked on Expasy´s ProtParam tool on the internet) of these peptides after a possible digestion at the hypothesized location of the β-casein. The first weight includes the N-terminal of the polypeptide to the Q (Glutamine) while the second polypeptide fragment includes the S (Serine) after digestion and downstream to the C-terminal. If TEV protease did, in fact, digest the β-casein at this position, there could be problems when using this protease in the future as a reliable tool for precise protein processing. However, to confirm these obtained results more extensive analysis would be needed. The activity of the recombinant TEV protease was later investigated using a synthetic polypeptide constructed specifically as a target for the protease. This synthetic polypeptide had an 8-time repeated amino acid sequence where each repetition ended in the recognition site for TEV protease and a histidine-tag at the very end. When running the SDS-PAGE and mass spectrometry assay with both an undigested polypeptide and a digested sample, a difference could be observed. A peak at roughly 2403 kDa could be observed in the digested polypeptide but not in the control. This peak corresponded well to the weight of one of the 8-time repeated peptides, indicating a successful cleavage of this part of the polypeptide. The peptide products were later analysed for bioactive properties and showed a dual-inhibiting function
(antihypertension and antidiabetic). This makes for a possible production of a multi-functional therapeutic agent in the future. (Less)
Please use this url to cite or link to this publication:
author
Nerjovaj, Fisnik LU
supervisor
organization
course
KBTM05 20241
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Tobacco Etch Virus protease, synthetic polypeptide, β-casein, UCSF Chimera, ApE (A Plasmid Editor), Escherichia coli, MBP (maltose-binding protein), SDS-PAGE, mass spectrometry, hypertension, type-2 diabetes, biotechnology
language
English
id
9165327
date added to LUP
2024-06-26 15:22:47
date last changed
2024-06-26 15:22:47
@misc{9165327,
  abstract     = {{Although different types of affinity tags have become a tool in the production and purification of recombinant proteins in various applications, the use of these have the risk of interfering with the biological activity and crystallization of proteins.There are both chemical and enzymatic ways to remove these tags, the enzymes include enteropeptidase, thrombokinase and protease to mention a few. The use of affinity tag-removing proteases has specifically proven to be useful. One of these enzymes include Tobacco Etch Virus (TEV) protease which recognizes the amino acids sequence ENLYFQ*G/S and cleaves between the Q and G/S, making it ideal for precise protein processing. Some limitations to these enzymes, like most other, are the fact that it has auto-cleaving properties which reduces the enzyme stability. In addition, the enzyme has solubility problems when it is produced by Escherichia coli. This can be circumvented by creating a fusion form of the protease with the chaperone MBP (maltose-binding-protein) which also prevents the auto-cleaving function. This thesis describes the production of a recombinant form of the Tobacco Etch Virus protease, analysis of the enzymatic reactions this enzyme has with different substrates and looks at various applications the obtained peptides can have. The substrates include a synthetic polypeptide created with the characteristic recognition site of TEV protease. The other substrate was β-casein which has a similar looking amino acid sequence compared to the recognition site for TEV protease. The methods leading this investigation included computational methods such as the visualisation and molecular modelling tool UCSF Chimera, where a docking could be achieved showing the interaction between the TEV protease and the substrates. Another computational tool included the use of A Plasmid Editor (ApE) program to construct the recombinant genes used for successful cultivation of the host organism E. coli and the expression genes for the produced polypeptides. The practical methods included a colorimetric assay with Folin-Ciocalteu’s reagent showing activity based on a reduction reaction led by reducing phenolic compounds in β-casein, SDS-PAGE and mass spectrometry analysis of the hydrolysed polypeptides. All results showed that TEV protease had activity and was not as specific as previously thought since a hydrolysis of the β-casein could be observed. The hydrolysis of the synthetic polypeptide could play a beneficial role in the future when manufacturing therapeutic agents since the peptides derived from the synthetic polypeptide after digestion have dual-inhibiting bioactivity against hypertension and type-2 diabetes.}},
  author       = {{Nerjovaj, Fisnik}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Production of recombinant TEV protease in Escherichia coli and analysis of enzymatic reactions}},
  year         = {{2024}},
}