Screening Techniques Using the Periplasmic Expression of Peptide Libraries and Target Molecules

Pharmaceutical antibodies have started replacing low-molecular weight pharmaceuticals, and now make up a large portion of the pharmaceutical market. Recently, middle molecular weight pharmaceuticals and peptide drugs are having attracted a lot of interest. Low-molecular weight pharmaceuticals are able to permeate cell membranes; hence, they can target intracellular markers. However, they also have low target specificity, making side-effects a concern. Pharmaceutical antibodies are highly specific to their targets, but it is currently difficult to design pharmaceutical antibodies that are able to permeate cell membranes. In contrast to low-molecular weight pharmaceuticals, which can be chemically synthesized, making production inexpensive, antibody drugs are produced in CHO cell cultures, making them more expensive. Peptide drugs have high target specificity, and drug delivery technologies allow them to permeate cell membranes. However, peptides are easily degraded by proteinases; therefore, pharmaceuticals need to be made degradation-resistant by chemical modification or other means. In spite of some difficulties, middle molecular weight pharmaceuticals and peptide drugs are world’s hope for the future. In this paper, several peptide display and related techniques are reviewed.


Introduction
As the population continues to age in developed country, the increasing number of patients and treatment costs are of great concern in the treatment of many different illnesses. In order to sustain healthy lifestyles and combat these illnesses, a current major focus in pharmaceutical research is the development of innovative new drugs that demonstrate increased therapeutic efficacy.
Pharmaceutical antibodies have started replacing low-molecular weight pharmaceuticals, and now make up a large portion of the pharmaceutical market. Recently, middle molecular weight pharmaceuticals and peptide drugs are having attracted a lot of interest. Low-molecular weight pharmaceuticals are able to permeate cell membranes; hence, they can target intracellular markers. However, they also have low target specificity, making side-effects a concern. Pharmaceutical antibodies are highly specific to their targets, but it is currently difficult to design pharmaceutical antibodies that are able to permeate cell membranes. In contrast to low-molecular weight pharmaceuticals, which can be chemically synthesized, making production inexpensive, antibody drugs are produced in CHO cell cultures, making them more expensive. Peptide drugs have high target specificity, and drug delivery technologies allow them to permeate cell membranes. However, peptides are easily degraded by proteinases; therefore, pharmaceuticals need to be made degradation-resistant by chemical modification or other means. In spite of some difficulties, middle molecular weight pharmaceuticals and peptide drugs are world's hope for the future. In this paper, several peptide display and related techniques are reviewed.
The target molecules of ICK peptides, which possess three intrapeptide disulfide bonds, are mainly membrane proteins (such as ion channels); they are generally considered to be suitable as templates for the development of peptide drugs [28,29].
GTx1-15, which used as an ICK peptide template, is stable in animal blood for multiple hours after intravenous administration without degradation by proteinases [30]. GTx1-15 is expected to have low immunogenicity like ziconotide which shows low immunogenic potential at animal models [31]. Optimization of the activity of ICK peptides is being conducted by the American companies Pfizer Inc. [32] and Amgen Inc. [33]. It is believed that ICK peptides can be valuable template peptides.

Target Molecules for Screening Periplasmic Expression
In this paper, membrane proteins used to refer to proteins that anchor in the cell membrane, and proteins in the cytoplasm are referred to as soluble proteins.

Membrane proteins
G-protein coupled receptors (GPCRs), ion channels, and transport proteins in the cell membrane are important pharmaceutical targets. The development of pharmaceuticals that act on GPCRs and similar proteins is ongoing [34], but that is not to say that pharmaceuticals that act on ion channels are necessarily being developed, especially for their ion channel characteristics. In order to shed light on this, PERISS method was performed, specifically targeting ion channels.
In general, it is thought that the expression of membrane proteins on E. coli inner membranes is difficult. Since only a small quantity of membrane protein can be expressed on E. coli inner membranes, techniques such as Western blot cannot detect the proteins. Thus, techniques for the direct measurement of the activity of the expressed membrane proteins on the E. coli inner membrane have been developed to allow the observation of the expression of small protein quantities that could not otherwise be detected [35]. When the E. coli cells are proliferating, inhibition of cell division by antibiotics results in long, rod-shaped E. coli, called "snake". By removing the outer membrane of these snake bacteria through techniques such as enzymatic treatment, giant spheroplasts with diameters of over 5 μm can be obtained. Through the application of patch-clamp electrophysiological techniques to these giant spheroplasts, the activity of ion channels expressed in the E. coli inner membrane can be directly measured. Successful measurement of expression using the patch clamp technique serves to confirm expression of the target ion channel on E. coli inner membranes. In addition to ion channels, some GPCRs are expressed on E. coli inner membranes. As detailed below, human type 2 muscarinic acetylcholine receptor has been used for the PERISS technique. If there is a high level of membrane protein expression, there will be many target molecules in the E. coli inner membrane; thus, screening with the PERISS technique will definitely be favorable. For increasing membrane protein expression on E. coli inner membranes, a chimaera membrane protein, which is fusion protein of membrane protein and maltose binding protein which is known as expression enhancer [36], or other expression enhancing protein, is synthesized. The chimaera membrane protein can achieve considerably higher expression of the target protein.

Soluble proteins
If the target molecule is a soluble protein, it will diffuse into the E. coli periplasmic space if it is expressed in the periplasm. However, the the formation of disulfide bridges; thus, peptides with multiple disulfide bonds can be expressed in their active state. For example, the camel antibody VHH, a so-called nanobody, can be expressed [19]. Using this technique, peptides from spiders and scorpions having 2 to 6 disulfide bonds within 17 to 76 amino acid residues showed a 60% success rate in the expression of the single correct isoform [20]. Chemical synthesis of peptides that contain three or more disulfide bonds is difficult; thus, a 60% success rate is considerable. Peptides obtained using E. coli periplasm peptide display techniques can be produced using the same periplasmic expression system. Usually, E. coli cannot perform secretory expression, but this has been achieved using a surfactant in the culture medium [JPN patent 5808529]. This technology holds potential for the industrialization of peptides with multiple disulfide bonds by periplasmic production.

Suitable Peptide Templates for Peptide Display
Many different peptides are used as templates for peptide display. For example, Fujii designs molecules with helix-loop-helix structures in a peptide library using artificial peptides as templates, and builds a peptide library by adding mutations to amino acids on the outer side, which do not affect the protein's three-dimensional structure [21]. This technology, which screens target molecules against a microantibody library presented by phage display, has been commercialized by Interprotein Inc. Meanwhile, Suga has developed a technique using a peptide library that includes special amino acids. In the RAPID system, which is based on the flexizyme system, a ring-shaped peptide library consisting of peptides approximately 15 amino acids long prepared with special amino acids has been created in vitro [22]. This technology has been commercialized by Peptidream Inc. Other peptide libraries are explained in detail by Kubo [23].
Another library different from the two peptide libraries stated above can be used-one that uses peptides found in toxins from various poisonous animals as templates. Animal toxins are promising pharmaceutical candidates because of their high target specificity. As an example, ziconotide, an inhibitor cysteine knot (ICK) peptide found in cone snails, is sold as a peptide drug that decreases pain experienced by patients with late-stage cancer. A peptide included in the three-finger scaffold of snake venom was used as a peptide library template because of its high specificity. An attempt was also made to create an anti-IL-6 peptide [11]. Over 30 new peptide candidates, including ICK peptides (GTx1,2), a peptide (GTx3) that is homologous with MIT1 from snake venom and Bv8 (a peptide discovered in frog skin), peptides homologous with peptides of unknown physiological functions (GTx4,5,6), a novel peptide (GTx7), and peptides homologous with translationally controlled tumor protein, cysteine-rich secretory protein, and venom allergen (GTx-TCTP, GTx-CRISP, GTx-VA) are discovered [24,25]. A common trait among these peptides is that they all have multiple cysteines in their peptide sequence, and form disulfide bonds. It has shown that the peptide GTx1-15 is voltage-dependent T-type calcium channel inhibitor [26]. GTx7-1 has an inhibitory effect on guinea pig isolated right atrial preparation [JPN patent 5019442]. Using these animal toxin peptides as templates can increase the likelihood of creating new peptide drugs. Companies such as Israel's Alamone Labs, France's Smartox Biotechnology, and Britain's Venomtech are all engaged in the research and development of pharmaceuticals based on natural toxins. The research and development of pharmaceuticals using animal toxin peptides is summarized well in the book Toxins and Drug Discovery [27]. integrated in tandem into a plasmid (Figure 1(1)). This allows both the target molecule and the peptide library to be expressed on a plasmid. The plasmid is transfected into E. coli, which expresses the peptide library in the periplasmic space, and expresses the target molecule on the E. coli inner membrane (Figure 1(2)). If the expressed target molecule is a membrane protein, it will be expressed on the E. coli inner membrane. In the case of a soluble protein, it is expressed as a fusion protein of a membrane protein and the soluble protein, so that the soluble protein is secured in the inner membrane, as explained in Section Soluble proteins. E. coli exhibits some incompatibility with plasmids; one E. coli cell can only retain one type of plasmid; thus, peptide expression in the periplasmic space is limited to one peptide from the library. Peptides expressed in the periplasmic space will bind to the target molecule if they have a strong binding affinity for it; if not, the target molecule will not bind to the peptide and will instead diffuse throughout the periplasmic space. The E. coli outer membrane is then removed enzymatically to form spheroplasts (Figure 1(3)). The peptide is expressed as a fusion protein with a tag sequence; hence, when the peptides that contain the tag sequence are concentrated using magnetic target molecule needs to be immobilized in the E. coli inner membrane for the PERISS screening technique. By immobilizing a soluble protein on the E. coli inner membrane as a membrane protein, it can then be treated as a target molecule. There are methods for immobilizing soluble proteins on the E. coli inner membrane that are similar to the APEx techniques described in Section E. coli periplasm peptide display techniques; however, instead of using these methods, a method is developing, wherein the soluble protein is fused onto the side of a membrane protein facing the periplasmic space. With this technique, either membrane proteins or soluble proteins can be used as target molecules for the PERISS technique.

Intra Periplasm Secretion and Selection (PERISS) Technique
Basic principles of the PERISS technique E. coli has both an outer membrane and an inner membrane, and the space between them is called the periplasm. In the PERISS technique, the target molecule DNA and peptide library DNA are 3) The E. coli outer membrane is removed and peptides that did not bind to the target molecule are washed away. The peptides that bound to the target molecule are collected by magnetic beads. 4) A portion of the peptide library on the plasmids in the E. coli is amplified by PCR, and once again integrated into plasmids. Steps 1-4 are repeated, and ultimately the amino acid sequence of a peptide that binds to the target molecule can be determined by analyzing the DNA sequence of the peptide library in the plasmids. M2 toxin is a three-finger scaffold venom peptide. Four amino acid residues in the middle finger structure, which were thought to play an important role in binding to the M2 receptor, were converted into a library, and the PERISS technique was conducted. As a result, the DNA sequence coding the original amino acid sequence was concentrated 333-fold in the peptide library ( Figure 2A) (manuscript in preparation).
The PERISS technique was also conducted with the tarantula venom, hanatoxin, using human potassium channel Kv2.1 as the target molecule. Hanatoxin binds to the voltage sensor domain on Kv2.1, which inhibits Kv2.1 activity. The four amino acid residues important in binding to the Kv2.1 sensor of membrane potential in hanatoxin were converted into a library, and the PERISS technique was conducted. As a result, the DNA sequence coding the original amino acid sequence was concentrated 1180-fold in the peptide library ( Figure 2B).

Pros and cons of the PERISS technique
In peptide display techniques other than PERISS, the peptide library and the target molecule must be prepared separately. When creating a peptide library for cell-free peptide display techniques, a separately prepared target molecule is added to a library and allowed beads, the target molecule is collected along with the E. coli. A part of the peptide library from the plasmid in E. coli is amplified by PCR, then integrated into the plasmid once again (Figure 1(4)); this process is repeated several times. Finally, I can analyze the DNA sequence from that part of the peptide library in the plasmid in E. coli, so that the amino acid sequence of peptides that bind to the target molecule can be determined. It is possible to monitor changes in the contents of the peptide library using next generation sequencing.
The process of spheroplast formation should be undertaken with particular care. Papers on the APEx technique make only brief mention of spheroplast formation, but in order to maintain favorable conditions for E. coli during spheroplast formation, one needs specialized knowledge regarding optical conditions for cell wall-degrading enzymes, such as buffer composition, temperature, reaction time, etc. If the reaction deviates from ideal conditions, it often results in the formation of what is called a "ghost, " where although it may appear that spheroplasts are formed, and the cytoplasm leaks out through a hole in the inner membrane. As discussed below, in the PERISS technique, plasmids in the E. coli cytoplasm are assumed to reproduce the information contained in DNA from a peptide library; ghost spheroplast formation reduces the effectiveness of the technique, and hence must be avoided.

Application of the PERISS technique
PERISS was conducted using a peptide library with tarantula venom to react. These techniques, therefore, have the advantage of being immediately available for use in the screening of low-molecular weight compounds. If the target molecule is a membrane protein, there is a benefit to screening cells expressing the membrane protein in culture, but the beneficial three-dimensional structure cannot be maintained when extracting the membrane protein from the cell membrane of the cultured cells, and many membrane proteins therefore lose their activity in the process. The surfactants needed to successfully extract a membrane protein from the cell membrane while preserving its activity differ based on the specific membrane protein; setting the conditions for this requires specialized knowledge. New technologies such as nanodiscs can be used, but if the target molecule is a membrane protein in vitro, these methods pose various anticipated difficulties. In the PERISS technique, membrane proteins are expressed on the E. coli inner membrane for screening; hence, there is no need to extract the membrane protein. Proteins such as ion channels that are expressed on the E. coli inner membrane can be directly observed electrophysiologically. There are no major differences compared to the previously reported basic characteristics of the expressed ion channels, but since the lipid composition of the inner membrane of E. coli, which is a prokaryote, differs from that of mammalian cells, the activities of peptides determined by the PERISS technique must then be re-evaluated in a eukaryotic system, such as by the patch-clamp technique using culture cells, and by Xenopus oocyte expression system using two electrode voltage clamp method. When creating a peptide library in E. coli by the PERISS technique, the library size is smaller than that of a cell-free system. By creating a library using an ICK peptide, which acts on ion channels, it is expect that there is a higher chance of including peptides that act on ion channels than in a random peptide library with no designated target molecule. This may allow the technique to overcome the disadvantage of a small library size. Furthermore, the small library size is made up for by creating multiple libraries, which can be done relatively inexpensively since the libraries are created using E. coli.
The PERISS technique has advantages and disadvantages, but it can complement other peptide display techniques and contribute to the field of peptide drug development.

Future Steps
The intra periplasm secretion and selection (PERISS) technique, a periplasmic peptide display technique in E. coli, can use various membrane proteins and soluble proteins as target molecules. Leveraging the characteristics of the periplasmic space and using animal toxin peptides with multiple disulfide bonds as peptide library templates, both increase the probability of peptides binding to the target molecule. Furthermore, the activity of the expressed membrane proteins in E. coli using PERISS can be measured electrophysiologically. Based on these three techniques and insights, membrane protein effective middle molecular weight pharmaceuticals and peptide drug research and development will go on.