The Effects of Self-cleavage Intein-ELK16 Tag in the Transcript Steric Hindrance of IFN

Abstract

Intervening proteins (Inteins) are identified as protein domains in a precursor protein structure. Inteins can excise itself from precursor protein and join the remaining portions which result in forming an active protein. In this study, the transcript expression level of recombinant human Interferon beta (rhIFNβ) connected to the self-cleavage Intein-ELK16 (LELELKLKLELELKLK) tag was measured by real-time PCR in HEK293T cell line. First, the sequence of Mycobacterium tuberculosis RecA (Mtu recA) was obtained from the InBase database to do appropriate changes including adding the restriction sites, kozak sequence, signal peptide and ELK16 sequence by SnapGene software. The RNA secondary structure were also examined using the online RNA Fold 2.2 web server. Next, the construct was inserted into pUC19 plasmid. The sequence of rhIFNβ was also cloned into pBudCE4.1 vector. In the next step, the rhIFNβ was ligated into the construct (self-cleavage tag of ELK16) using T4 DNA ligase and the recombinant construct was transfected into HEK293T cell line. Finally, expression of the cassette was evaluated by real-time PCR. The analysis of secondary RNA structure indicates a minimum free energy of MEF − 261.10 kcal/mol. Our results indicate that IFNβ was upregulated (37.8-fold, p < 0.0001) in cells which transfected by rhIFNβ-ELK16 compared to the mock and un-transfected conditions. Altogether, our results show that the presence of mini self-cleavage Intein-ELK16 tag along with the rhIFNβ had no interference in transcription of rhIFNβ in the HEK293T cell line.

Introduction

Intervening proteins (Inteins) are internal protein domains, placing in a precursor protein. Inteins play important roles in the post-translational process with the ability of self-excision. It causes splicing of the flanking polypeptide sequences called external proteins (exteins). This post-translational process is a spontaneous autocatalytic process which known as protein splicing [1–5]. Since the discovery of the first yeast intein (called VMA1 protein (in the late 1980s, more than 500 inteins have been identified in 200 species of microorganisms [2, 6]. Inteins play different functions and most of them are involved in the replication system [6]. Most of the inteins contains 350–500 amino acids in length, including an endonuclease domain (cis-splicing inteins). This domain can be eliminated without a negative effect on the function of the intein [7]. Inteins are valuable and attractive biotechnological tools in a wide range of biotechnological usages such as protein purification and specific modifications. Because of their unique chemical reactions, they don’t need additional requirements and they can independently splice in a wide variety of exteins [3, 8–10]. Using targeted mutation in specific residues neighbor of splice junctions, self-cleaving inteins are generated and they can be induced by pH or dithiotheritol (DTT) reagent [3, 11]. The application of self-cleaving inteins and affinity tags such as maltose-binding protein (MDB) or chitin-binding domain (CBD) results in a new cost-effective protein purification system [11–13]. However, the main problem is their early pre-cleavages in vivo, effecting on the final yield of the purified protein. This disadvantage has reduced their popularity and demand for the large-scale application. Most of the Inteins, as single continuous polypeptides (cis-splicing inteins), are produced in a single polypeptide form. But, some of them are naturally transcribed and translated as split intein in two or more polypeptide parts such as DnaE intein or artificial split intein, such as split intein derived from the Mycobacterium tuberculosis RecA (Mtu RecA) intein. Split inteins are separately translated and assembled spontaneously to initiate protein trans-splicing (PTS) [4, 14, 15]. It seems that protein trans-splicing (PTS) using split inteins in protein purification systems resolve the premature cleavage problem during the expression of recombinant protein in vivo [16].

There are few short self-assembling peptides such as ELK16 (LELELKLKLELELKLK), 18A and L6KD which induce the protein aggregation in the fusion of different target proteins. Especially, the ELK16 is a β-sheet-self-assembling peptide which can use as “pull-down” fusion tags in the purification protein system without any chromatography step [17–19]. In this study, we assessed the transcript expression level of recombinant human Interferon beta (rhIFNβ) conjugated to the self-cleavage Intein-ELK16 tag using computational and experimental procedures. In fact, the steric hindrance of presence of self-cleavage Intein- ELK16 tag linked to rhIFNβ was technically evaluated at the mRNA expression level by real-time PCR.

Materials and Methods

Plasmid Construction

To digest and ligate DNA fragments, KpnI, BglII, Eco31I restriction enzymes and T4 DNA ligase were used (Thermo Fisher Scientific.co). I_c (ELK16-Intein C-terminal) construct was respectively included from 5′ to 3′: KpnI restriction site, Kozak sequence, signal peptide, ELK16, PT linker, I_c (the last 58 amino acids of ΔI-CM intein) (mini Intein Cleavage Mutant) and Eco31I restriction site (Fig. 1). Also I_N (ELK16-Intein N-terminal) was respectively included from 5′ to 3′: KpnI restriction site, Kozak sequence, signal peptide nucleotide sequence, ELK16 nucleotide sequence, PT linker, I_N (the nucleotide sequence of the initial 110 amino acids of ΔI-CM intein) and BglII restriction site. In addition, the amino acid sequence was converted to nucleotide by the SnapGene software (GSL Biotech; snapgene.com) and nucleic acid codon optimization was done for appropriate expression in eukaryotic cells. The secondary RNA structure was examined using the online RNA Fold 2.2 web server [20]. Then, the constructs were synthesized by Sinaclon Company. The pUC19 plasmid backbone was digested with KpnI and Eco31I enzymes (pUC19 I_C) and with KpnI and BglII enzymes (pUC19 I_N) to create the sticky ends. The pBudCE4.1 IFNβ plasmid was previously cloned by Norouzi et al. [21]. To earn the interferon sequence of plasmid and introduce the restriction cutting site of Eco31I and BglII at the end of the insert, appropriate primers were designed by Oligo 7 software (F: 5′-ATT GGT CTC CCA ACA ATG AGC TAC AAC-3 and R: 5′-GAA GAT CTT CTC AGT TTC GGA GGT AAC C-3′) (The bold nucleotides in the forward and reverse primers are Eco31I and BglII restriction sites). Then, the PCR was performed in a 25 μl reaction mixture containing Taq DNA Polymerase 2× Master Mix RED 1×, 120–160 ng/µL template, IFNβ F primer 0.4 mM, IFNβ R primer 0.4 mM, ddH2O Up to 25 μL. The pBudCE4.1 plasmid was also linearized by KpnI and BglII enzymes.

Fig. 1.

Protein splicing and Schematic view of the designed construct: a Protein splicing: The Intein DNA sequence is transcribed into the mRNA and translated into a precursor protein, and then, with the release of the intein and the binding of two pieces of extein, they become a mature and active protein. b Schematic view (designed as illustrated by Gene Designer) on how to locate the sequences in construct (ELK16-Intein tag)

The I_C construct was digested with KpnI and Eco31I restriction enzymes. In addition, the I_N and the pBudCE4.1 IFNβ plasmid were digested with KpnI and BglII restriction. The IFNβ gene digested with Eco31I and BglII restriction enzymes. Then, the digested I_C (with KpnI and Eco31I) and the digested rhIFNβ (with Eco31I and BglII) fragments containing the overlapping Eco31I cutting site were ligated. Finally, the ligated fragment (Ic plus rhIFNβ) was inserted between the KpnI and BglII sites of pBudCE4.1 (Fig. 2a) which results in pBudI_C-rhIFNβ expression plasmid construct (Fig. 2b). The digested I_N fragment was also ligated into pBudCE4.1 plasmid between the KpnI and BglII sites to make the pBud I_N expression plasmid (Fig. 2c). Afterwards, the construct was sequenced by Sinaclon Co. The final construct was used for transfection of HEK293T cell line.

Fig. 2.

Schematic view of the original plasmid and recombinant constructs: a there are two multiple-cloning sites on the pBudCE4.1 vector, one is located at the downstream of the EF-1α promoter, and the other one is located in the downstream of the CMV promoter. b The ligated fragment (Ic plus rhIFNβ which is shown in blue rectangle) is inserted between the KpnI and BglII restriction sites on pBudCE4.1 framework, which results in recombinant of pBudIc-rhIFNβ expression plasmid construct. c The digested I_N fragment was inserted into pBudCE4.1 framework plasmid, between the KpnI and BglII restriction site (which is shown in green rectangle) that results in recombinant pBud-I_N expression plasmid construct

Transfection of HEK293T Cell Line by Recombinant pBud I_c-IFN and pBud I_N Plasmids

The HEK293T cell line was purchased from the Pasteur Institute, Iran. The constructs (Table 1) were transfected into HEK293T cell line with the Ca-phosphate method [22]. In this method, CaCl2 2.5 M was utilized to form the phosphate and DNA cluster complex. The 2X HBS (50 mM HEPES, NaCl2 80 mM, 1.5 mM Na2HPO4) was also added to the mixture and incubated at 37 °C with 5% CO2 for 24 h in a complete medium (DMEM, High Glucose, GlutaMAX + 10% FBS + P/S 1%). The media were then exchanged by a fresh medium containing and incubated for a further 24 h.

Table 1.

Plasmids used for transfection of HEK293T cell line

Construct	Description
pBudIC-rhIFN	The pBudCE4.1 contain the main structure for evaluating the effect of tag on the expression of interferon
pBudIN	The pBudCE4.1 contain N terminal of intein as control
pBudCE4.1	The pBudCE4.1 without insert as mock control
pBud-VGF	The pBudCE4.1 with different insert as scramble control
Untransfected HEK293T	For control and comparison

Expression Analysis of rhIFNβ

After 72 h from transfection, RNA was extracted by the Trizol method (Invitrogen USA) according to the manufacturer’s protocol. Extracted RNA was treated with DNaseI to remove genomics DNA. 2 μg of the extracted RNA was converted to cDNA using the cDNA synthesis kit (Thermo Fisher Scientific. Co). Finally, in order to evaluate the expression of rhIFNβ in the HEK293T cell line, real-time PCR was performed by SYBR Green from TAKARA (SYBRPremix Ex Taq (2X) (Tli RNaseH Plus), ROX plus (TAKARA Bio INC) using the specific rhIFNβ primers (IFNβF: 5′-GAC AGG ATG AAC TTT GAC-3′, IFNβR5′-ATA GAC ATT AGC CAG GAG-3′) and the specific eEf1a1 primers (eEf1a1F: 5′-CCCTTCTGGCTTACACACT3′, eEf1a1R: 5′-TGA ACC AAG GCA TGT TAG CAC-3′). The reaction was performed using Maxima SYBR Green/ROX qPCR Master Mix (2X) 1x, F primer 0.4 pmol/μL, R primer 0.4 pmol/μL, Template cDNA 8–12 ng/μL, ddH2O up to 25 μL.

Statistical Analysis

Statistical analysis was performed via GraphPad.Prism.v7.03 through non-parametric tests.

Results

Intein-ELK16 self-cleavage tag sequences were designed and optimized for the expression in eukaryotic HEK293T cells. First, insilico studies was shown a secondary structure with a minimum free energy (MFE) − 261.10 kcal/mol (Fig. 3), and then constructs synthesized by Sinaclon Company, which was prepared within plasmid pUC19 (pUC19 I_C and pUC19 I_N). The pUC19 I_C plasmid (containing the ELK16-I_C) and pUC19 I_N (containing the ELK16-I_N) were transformed, large number of colonies were grow and visible on the selective medium (Fig. 4a). To confirm the transformation, extracted plasmids were separated by electrophoresis on agarose 0.7% with 1 Kb DNA ladder (Thermo Scientific, USA) and bands with 3000 bp in size were observed (Fig. 4b). In order to confirm the presence of the I_C (ELK16-I_C), double enzymatic digestion was performed on extracted plasmid and the 374 bp fragment was exited from the plasmid (Fig. 4c). The amplified rhIFNβ inserted into TA vector, was 530 bp in length on 1% agarose with 1 Kb DNA ladder (Thermo Scientific, USA) (Fig. 4d). Also, the result of sequencing showed that ELK16-I_C tag and rhIFNβ were correctly ligated in the pBudCE4.1 plasmid (Fig. 5). Afterwards, mRNA expression analysis by real-time PCR was demonstrated approximately 38-fold elevated (The calculating formula based on the 2^−ΔΔCt) expression of rhIFNβ in comparison with un-transfected and mock vector and statistical significant (p < 0.0001). (Figure 6).

Fig. 3.

RNA secondary structure results by RNAFold. a RNA secondary structure with minimum free energy between the pairs of base (MEF of − 261.10 kcal/mol). b A mountain plot represented the MFE structure, the thermodynamic ensemble of RNA structures, and the centroid structure (the red, green and blue lines referred to the minimum energy of the free energy (MFE), the probability of the pair (PF) and the centroid algorithm, respectively). Additionally, the positional entropy was represented below

Fig. 4.

A summary of the construction procedure for attachment of the self-cleavage Intein- ELK16 tag to Interferon β: (A) E.coli Top10 competent cells after transformation of pUC19 plasmid containing I_C (self-cleavage Intein- ELK16 tag), colonies were appearance on the LB agar plates containing the ampicillin (100 μg/mL). (B) The extracted plasmids of pUC19 I_C from transformed colonies, separated by electrophoresis on 0.7% agarose gel. Lane 1: 1 Kb DNA ladder (Thermo Scientific, USA, Cat No. SM0313), lanes 2 and 3: pUC19 I_C plasmid (~ 3060 bp). (C) The Results of enzymatic digestion of pUC19 I_C by KpnI and Eco31I loaded on 1% agarose gel. lane 1: 1 Kb DNA ladder, lane 2: undigested pUC19 I_C, lanes 3 and 4: Double digested pUC19 I_C (There are two restriction site for Eco31I on the pUC19 I_C vector) I_C Insert 374 bp and pUC19 digested ~ 1330 bp, lane 5: pUC19 I_C Mono digestion with KpnI, ~ 3060 bp. (D) The results of enzymatic digestion by BglII and Eco31I enzymes on TA vector containing rhIFNβ (as insert) separated by electrophoresis on 1% agarose gel. lane 1: 1 Kb DNA ladder, lane 2: undigested TA- rhIFNβ, lane 3: Eco31I mono digestion, there are two bands ~ 2050 bp and ~ 1360 bp (Because there are two Eco31I restriction sites on the TA- rhIFNβ plasmid, so there are two bands on the gel.), lane 4: BglII mono digestion, lane 5: Eco31I and BglII digestion, there are three bands on the gel ~ 1520 bp, ~ 1360 bp and ~ 374 bp (insert)

Fig. 5.

Confirmation of the structure of Interferon β linked to self-cleavage Intein- ELK16 tag by sequencing

Fig. 6.

Enhancement of the expression of Interferon β linked to self-cleavage Intein- ELK16 tag in HEK 293T cell line. An elevated amount of Interferon β expression is seen in the transfected cells in comparison with un-transfected cells and controls, [approximately 37.8-fold change p < 0.0001 (one way Anova, multiple comparison)]. This Data are presented as mean ± SD of duplicate experiments (Graph Pad Prism 7). Asterisk (****) indicates statistical significance

Discussion

Nowadays, the production of many therapeutic proteins such as rhIFNβ, insulin and coagulation factors is possible by recombinant genetic engineering in the host cells such as prokaryotic and eukaryotic cells. Since a wide variety of the proteins are expressed in the host cells, the proteins of interest should be purified with high purity and quality from undesirable proteins. On the other hand, the common affinity chromatography methods and their instruments are costly, time-consuming and needed to expert technicians [23]. Accordingly, the requirement of the pharmaceutical industry for a low-cost and rapid method to purify recombinant proteins is vital [24]. Inteins are the mobile genetic elements in the host protein, translated into the proteins, including functional roles only at the protein level [13]. The ability of connecting the intein to a target protein has greatly expanded their application. They can be used in conjunction with an affinity group to purify a desired protein [25]. Genetic modification by suitable mutations in the structure of the inteins causes breaking down and it can be used as an alternative to protease digestion in the protein purification system. Mtu RecA (recombinase enzyme of M. tuberculosis) is separated into two parts by intein sequence [26]. The Mtu RecA is composed of 440 amino acids, including protein splicing and homing endonuclease parts. The engineered Mtu RecA (ΔI-CM) is a mini and continuous intein (168 amino acids) containing 110 amino acids of N terminal and 58 amino acids of C terminal of the integrative Mtu RecA. It was constructed by the removal of the homing endonuclease domain which result in a reduction in activity [26, 27]. To restore activity at the same level of the full-length intein, two mutations including, V67L (ΔI-SM, splicing mutant) and D422G (ΔI-CM, cleaving mutant) were made [26]. Meanwhile, split inteins are divided into two parts: the N-intein and C-intein. Separated parts do not have any activity, but they can interconnect with each other and cause trans-splicing of protein in contiguous form [24]. A number of inteins, such as DnaE split intein, are naturally split, but mutations and changes in the critical amino acids of DnaE split intein cause reduced their performance. However, there are artificial inteins which were formed by the manipulation of continuous intein. The functions of these artificially split parts are then the same as the natural split inteins [7, 28]. The Mtu Rice (ΔI-CM) is split in two parts including N terminal and C terminal, each contains 110 and 58 amino acids, respectively [11]. To prevent early cleavage, each of these sections (N terminal and C terminal) is kept initially separated from each other (by performing two independent transfections). When they re-associate in vitro, they cause trans-splicing of the target protein. ELK16 is a small peptide which can create hydrophobic beta structures and spontaneously accumulate due to the combination of its amino acid. ELK16 keeps its properties when it is attached to another protein and therefore, can be used as a replacement for chromatography columns in a protein purification system [19]. Over the past few years, attempts to use of self-cleavage tag based on mini intein and self-aggregating peptides have provided hope, that expensive proteases and the chromatography column would be removed from the protein isolation procedure. This will then introduce a cheap, fast and efficient protein purification system. The main obstacle for such a system would be the impact of these extra parts on the expression of the main mRNA. The final purified protein would be fortunately intact, so there is no concern about the structure and the activity of the final protein product.

In this research, the purification tag was designed in such away to be used for separation and purification of recombinant protein from other host proteins. The tag includes ELK16 self-assembling peptide and self-cleavage split intein Mtu RecA (ΔI-CM). Each of the N terminal and the C terminal of split intein Mtu RecA (ΔI-CM) connects to an ELK16 peptide (for their precipitation). These extra parts that were added to the native rhIFNβ mRNA may have also some sort of impact on the stability of mRNA too. In order to study the effect of these tags on the transcript expression level, the expression of rhIFNβ in conjunction with self-cleavage Intein- ELK16 tag was measured in the transfected HEK293T cell line using the real-time PCR. The used ELK16 is a small piece of amino acids. It can be easily aggregated with centrifugation at 4 °C along with the target protein. Mixing the sediments from two separate transfected cells (N terminal fragment and C terminal fragment of Mtu recA (ΔI-CM)) and changing the pH (by cleavage buffer: PBS supplemented with 40 mM Tris-Bis, pH 6.2, 2 mM EDTA), the intein may activate and the target protein may release in the solution buffer. Another advantage of the construct is the presence of the restriction site for the Eco31I (BsaI) enzyme on the C terminal of tag. The BsaI enzyme is a member of type IIs restriction enzymes and therefore its recognition site is different from its cut site. This enzyme may be able to connect different types of target proteins to the C-terminus. After the digestion of the I_C construct and the rhIFNβ (target sequence) by BsaI, it is expected that they ligated together without any nucleotide deletion or insertion. Then, the final construct will be placed in prokaryotic or eukaryotic expression plasmids. Shi et al. [11] used the combination of self-cleavage split Intein Mtu RecA (ΔI-CM) with elastin-like polypeptide (ELP) in order to purify recombinant protein. ELPs are artificial peptide polymers derived from the hydrophobic domain of tropoelastin containing 20–330 repeated VPGXG pentapeptide, where X can be any amino acid except for proline [29, 30]. The large size of the ELP could be a disadvantage point compared with the small size of ELK16. The ELP also needs a precise temperature control called Inverse Transition Cycling [30]. Wu et al. [19] purified the target proteins using ELK16 tag. Since the ELK16 had not been used with Intein, an enzymatic digestion was required for releasing the target protein of ELK16. Therefore, this process was not only costly but also time consuming. Indeed, in this study for the first time, the ELK16 peptide and the self-cleavage split Intein Mtu recA (ΔI-CM) were simultaneously used in the structure of a construct for purification of the recombinant protein. So, the target protein attached to the ELK16 may easily be accumulated and precipitated by centrifugation, by self-cleavage split Intein Mtu recA (ΔI-CM). It may be able to detach and purify the target protein as a fast and cost–benefit method. Furthermore, the kozak sequence and the signal peptide were also considered and added to the final construct. They not only elevate the protein production (kozak sequence), but also facilitate the secretion of the recombinant protein in eukaryotic cells.

Since the RNA molecule plays a very important role in the expression pathway, the prediction of its secondary structure for cellular and molecular studies is of great importance. In this regard, calculating the minimum free energy (MFE) based on similarities in the stable motifs of RNAs is the most common method for predicting the secondary structure of a single-strand RNA. MEF is the sum of the free energy between the base pairings that participate in the formation of the secondary structure. Therefore, the most negative value means the more stable structure. Based on the in silico study, the final structure was investigated to determine the secondary structure of RNA by RNAfold web server. The results showed that, based on the total free energy between the base pairs, a secondary loop-ring structure was created at 37 °C with a minimum free energy of MFE of − 261.10 kcal/mol (Fig. 3). Experimentally, self-cleavage Intein-ELK16 tag was successfully designed and cloned. The recombinant construct in which rhIFNβ connected to self-cleavage Intein-ELK16 tag in the pBudCE4.1, was finally transfected into the HEK293T cell line. The expression of rhIFNβ in HEK293T cell line was compared with un-transfected (as a negative control) and mock using real-time PCR. For normalization the qPCR data, the eEf1α1 housekeeping gene was used as the internal control.

Conclusion

In conclusion, our results indicate that the self-cleavage Intein-ELK16 tag had no inhibitory effect on the mRNA expression level of the rhIFNβ. We have also concluded that those extra parts have no instability effect on rhIFNβ mRNA (a huge amount of mRNA was detected). This result could be implied that the self-cleavage Intein-ELK16 tag did not exert the steric hindrance on the transcript expression of rhIFNβ and most likely any other mRNA which should validate through further studies. The suggested tag might be used as an alternative to the chromatography purification system which can eliminate enzymatic reaction by easy-going procedure.

Funding

This study was funded by Iran National Science Foundation (Grant No. 92042856).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.