Intein-mediated purification system: mechanism and applications

Sarra setrerrahmene　Shuhua Tan

Abstract：The incorporation of self-cleaving protein elements into a variety of fusion-based purification systems; has been an important development in the area of recombinant protein purification. The self-cleaving capability of these tags has recently been combined with additional purification tags to generate novel and convenient protein purification methods. This review elucidates the properties of intein, the mechanism of the intein-based protein splicing and the progress of intein-based protein purification procedures, and recent advances in the applications of intein.

Keywords: self-cleaving; fusion; protein purification; intein.

Introduction

A major benefit resulting from the advent of recombinant DNA technology has been the large-scale production of proteins of medical or industrial importance. While production of heterologous proteins in bacterial hosts has been implemented successfully in the biotechnology industry; the large variability of recombinant protein in their expression, solubility, stability, and functionality, makes a difficulty for their large-scale analyses and production.

When expressing and purifying large quantities of soluble protein, expression difficulties often include poor yield and the formation of insoluble aggregates, consequently, advances in recombinant protein expression tried to find solutions for these problems, starting by the development of better expression systems and host strains, improving mRNA stability, host-specific codon optimization, arriving to the use of secretory pathways, post-translational modification, co-expression with chaperones, and decreasing the amount of proteolytic degradation. However, no other technology has been as effective in improving the expression, solubility, and production of biologically active proteins as the addition of fusion tags, especially for difficult-to-express proteins.

Genetically engineered fusion tags can be defined as exogenous amino acid sequences with a high affinity for a specific biological or chemical ligand (1;2).they allow the purification of virtually any protein without any prior knowledge of its biochemical properties. They can improve the variable yield and poor solubility of many recombinant proteins. On the other hand, adding fusion tags has been reported to result in changes in protein conformation, poor yields, loss or alteration of biological activity, and toxicity of the target protein. For this reason, it is desirable to remove the tag from the target protein after expression (3; 4). To enable removal of the tag, a linker region is typically included between the tag and the native protein sequence.

By using a self-cleaving intein affinity tag in protein purification, the need for proteolytic cleavage of purified fusion proteins can be eliminated thus enabling purification of native recombinant protein in a single chromatographic step, the structure and properties of this self-cleaving element have been investigated by a number of research groups. Successful studies have now established that this splicing element is academically and industrially valuable in protein purification.

Definition

The term” Inteins” is derived from ‘INTervening protEINS, because they are intervening sequences embedded in a host protein precursor sequence; inteins are capable of post-translational self-excision from a host-intein precursor protein through a process known as "protein splicing" (5).

Inteins are genetically similar to self-splicing introns, and both can self-excise from the precursor sequences. In contrast to introns which excise themselves at the precursor RNA level , inteins are transcribed and translated together with their host protein and only at the protein level they excise themselves from the host protein. The two portions of the host protein separated by the intein are called exteins (6).

From the discovering of the first intein in 1987 until now, over 350 inteins have been identified in a wide variety of proteins from bacteria and archaea to eukaryotes.14 most known inteins are confined to DNA metabolic functions or pathways. These include DNA polymerase, helicases, gyrases, RecA recombinase, ribonucleotide reductases, among others. Inteins generally share low sequence similarity, but have a high degree of similarity in structure, self-excision mechanism, and evolution.

Intein structure

All inteins consist of three domains (7): two self-splicing (N- and C-terminus domains) and an endonuclease domain play a crucial role in the spread of inteins. These are known as large inteins. The mini-inteins are formed only by the two self-splicing domains:

1. The N-terminal splicing domain: formed by the 2 conserved motif A and B and two additional motifs characterized by Pietrokovski (N2 and N4)

1.1. Motif A: is the N end splice junction, it contains the chemically essential Ser or Cys residue (8). 1.2. Motif B: protein splicing suggests an N -- 0 acyl shift of serine or threonine residues at the splice sites with the assistance of a histidine residue, and suggests the conserved histidine at the C splice junction . The seventh residue in motif B is invariably a histidine and might fulfill the required function.

2. The C-terminal splicing domain: ends at the aa following the C end of the inteins. The C- terminal splice junction area is composed of two motifs (F and G) that are either consecutive or separated by one or two aa. 2.1. Motif F: contains an aromatic residue on both sides of several acidic and hydrophobic residues. 2.2. Motif G: is characterized by the three conserved C-terminal splice junction residues preceded by four hydrophobic residues and contains the first extein residue following the intein.

3. The DOD endonuclease: The endonuclease activity involves the central blocks C, D, E, and H Blocks C and E are the dodecapeptide motifs required for endonuclease activity. 3.1. Motif D contains the conserved basic amino acid Lys and a Pro residue, and maintains the distance between blocks C and E 3.2. Motif H, composed by19 amino acid motif, found between blocks E and F Block H is characterized by one or more Ser or Thr residues in positions 1–3, a central hydrophobic region containing several Leu and a Gly at position 18 followed by ahydrophobic residue.

In fact, only one His in Block B, two Gly in Block C (excluding inteins lacking this block) and one Asn in Block G are present in all inteins (Figure1)

Figure 1: large and mini-intein structure

Mechanism of intein-based protein splicing

Protein splicing is a post-translational processing event. The mechanism of splicing is now very well established and understood (9). Intein-mediated protein splicing pathway consists of four nucleophilic displacement reaction steps, coordinately arranged in vivo (10). Resulting in peptide bond cleavage at both intein–extein splice junctions, and ligation of the flanking sequences to yield a mature extein and an excised intein (11;12). The four displacement reaction steps are:

1. Activation of the N-terminus of the intein by an N-O shift for serine or N-S shift for the cysteinethat leads to an ester or thioester intermediate. This rearrangement leads to the N-extein binding to the oxygen of a serine or to the sulfur of a cysteine residue at the splice junction.

2. Transesterification between the ester or thioester on intein N-terminus and the nucleophilic residue of the C-extein, this reaction generates a branched protein intermediate.

3. peptide bond cleavage and excision of the intein, this cleavage occur via an aminosuccinimide intermediate for the intein having an asparagine residues at their C-terminus; or via an aminoglutarimide for intein posecing an glutamine terminus (13).

4. spontaneous S-Nacyl rearrangement of the ligation product from the ester or thioester to a stable amide bond between the two exteins. These 4 steps of protein splicing are so rapid that the precursor protein is rarely observed.

most inteins begin with Cys or Ser, and end with His-Asn, while some inteins begin with Ala, Gln, and Pro or end with Asp and Gln. C- exteins usually begin with Cys, Ser or Thr. These conserved splice junction residues are directly involved in protein splicing mechanism .substitution of Asp for the Gln which resides at the C-terminus of some inteins can moderately improve the rate and extent of protein splicing(14). However, when the penultimate His is mutated to Ala, protein splicing is prevented.This indicates the important role of the conserved penultimate His residue in Asn cyclization.it has been showed that this histidine serves as a proton donor to the carbonyl oxygen of the terminal Asn (15; 16).

Application of inteins in biotechnology

Recombinant protein purification has benefited lot from the applications of protein splicing. Any desired gene can be cloned to the N or C terminus of an intein gene, and expressed in frame to the intein tag (17). The creation of self-cleaving protein elements that can be combined with conventional affinity column has generated an effective self-cleaving affinity tags which have the ability to release a target protein fused (18; 19) Either C or N-terminally to the tag, in response to a simple chemical or physical stimulus. The specificity of the cleaving reaction allows the affinity tag to be removed without the addition of expensive protease and prevents unwanted cleaving. On other hand, the cleaving reaction can be induced while the tagged target is bound to the affinity column, thus eliminating the need for other step to remove the cleaved tag. To facilitate the release of the recombinant protein from the intein and prevent its modification by the reducing agent, random mutation of naturally intein has been done to select a mini-intein with pH-sensitive C-terminal cleavage. These mini-inteins inteins have been incorporated into a commercial purification kit (IMPACT-TWINTM, NewEngland Biolabs).

This IMPACT-TWINTM use a mutated intein (substitution of Asn454 with Ala), which exhibites N-terminal cleavage at the presence of 1,4-dithiothreitol (DTT) orβ-mercaptoethanol at low temperatures and over a broad pH range (5.5–9.0). The C-terminus was combined with a chitin binding domain to make protein purification simpler and more convenient than the conventional purification system (20). Chitin binding domain (CBD) was the first used affinity tag, later other tags have been developed such as ELP (21), cellulosebinding domain(22), polyhistidine(23),glutathione S-transferase ,FLAG-tag(24), and maltose-binding protein .Using this purification system the whole process could be completed within 2 days. The final purity of target protein was in general more than 95% and the yield was similar to the yield from conventional affinity purification methods , studies have also shown that the bioactivities of the target proteins were identical to those isolated using the conventional procedures(25;26).

Despite its distinct advantages, this purification method still has several problems, such as relatively low yield and low reproducibility of the protocol. The intein-based purification system is still limited to laboratory scale because of the high cost of the affinity matrices (27; 28). In addition to the application in protein purification, the generation of greenfluorescent protein mini-intein fusion system has simplified the process of optimizing the expression of fusion by direct correlation between the cell fluorescence level and protein yield (29).The green fluorescent protein has also been used as a reporter system for protein-protein interactions, and high-throughput drug screening. On other hand, Intein expression system has been successfully applied in cytotoxic proteins synthesis. The cytotoxic protein is inactivated in vivo by its fusion to an intein, and the pH-controllable splicing of intein is proceeded in vitro to liberate the active cytotoxic protein. this technic has been succefuly used to express the cytotoc proteinI-Tev in E.coli .Intein systeme has been also used by Daugelat & Jacobs (30) in epitope mapping and antigen screening.

Conclusion

The remarkable self-cleaving property of intein can have numerous novel applications in downstream processing; with the intein-mediated fusion protein production system, a protein with an affinity tag can be purified in a single chromatographic step.

In this paper we have provided an overall picture of the principles, characteristics, and mechanisms of various intein systems and their potential applications in downstream processing, especially protein expression and purification. We expect that it will be a useful contribution for researchers interested in the intein system.

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Unit: State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China.

Corresponding author:

Shuhua Tan, Ph.D. State Key Laboratory of Natural Medicines China Pharmaceutical university .Nanjing 210009. E-mail: tohike@hotmail.com.Tel: 86-25-83271012