Simple and rapid pipeline for the production of cyclic and ...

Highlights

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  A novel HOE tag was designed.

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  Small-sized peptides fused with the HOE tag were produced in recombinant form.

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  The tertiary structure of the peptides was preserved.

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  The tag was cleaved both chemically and enzymatically.

Abstract

Small and medium-sized peptides are gaining popularity in biomedical applications, including therapeutic target development. As an alternative to chemical synthesis, we describe a complete pipeline for the production of linear as well as structurally constrained cyclic peptides in an E. coli expression system in this study. A plasmid vector containing a novel N terminal HOE tag (28 amino acids in length) that fuses with the peptide was created. The HOE tag contains sites for both chemical (CNBr) and enzymatic (enterokinase) cleavage, making it easy to isolate the peptide after production. A total of 21 peptides (17 cyclic and 4 linear) were synthesized, and the HOE tag was successfully removed using either CNBr (9 peptides) or enterokinase (12 peptides). The presence of a disulfide bond was confirmed in six representative cyclic peptides. In this study we have provided detailed instructions on primers design strategy, overexpression and purification of HOE tagged peptides, chemical and enzymatic cleavage, and confirmation of the cyclic form of peptides. We are confident that this pipeline will assist researchers in producing multiple recombinant peptides in a cost-effective and time-efficient manner.

Introduction

For several decades, there has been an exponential increase in the use of bioactive peptides for diagnosis and therapy. Peptide-based drugs are excellent therapeutic candidates due to their specificity, efficacy, and low toxicity. To name a few, there are more than 150 FDA-approved peptide-based medicines on the market, which are used to treat cancer, metabolic and hematological disorders, diabetes, hypotension, osteoporosis, and HIV [1]. The global market for peptide-based drugs was valued at USD 25.35 billion in 2018, and with a 9% annual increase, it is expected to reach USD 50.60 billion by 2026 [2].

George P. Smith's discovery of phage display resulted in a new scientific breakthrough in peptide-based drug development [3]. The development of a combinatorial phage display peptide library containing random peptides displayed on the phage virion has opened up new avenues for peptide research, drug development, and high-throughput protein interaction screening. As a result, several peptides discovered through combinatorial phage display peptide library screening have entered clinical trials to treat viral infections and cancers [4,5]. As commercially available phage display peptide libraries, New England Biolabs offers randomized heptapeptide (Ph.D.-7), dodecapeptide (Ph.D-12), and loop constrained heptapeptide (Ph.D-C7C). The Ph.D.-7 and Ph.D.-12 libraries contain linear peptides, whereas the Ph.D.-C7C library contains 7-mer peptides arranged in the shape of a loop due to the disulfide linkage of oxidized cysteine residues flanking the peptide segment. Despite a plethora of scientific publications describing the efficacy of peptides as therapeutics, biotechnological production of small-sized peptides remains a challenge [[6], [7], [8], [9], [10], [11], [12]].

Peptides are typically synthesized chemically or using recombinant DNA technology [13,14]. The chemical synthesis of peptides used by the majority of manufacturers is accomplished through solution-phase synthesis (SPS), solid-phase peptide synthesis (SPPS), or a combination of both [14]. Even though commercial suppliers can produce peptides chemically on a large scale, it is expensive (500 €/peptide) and can take a long time (>6 months). Peptide synthesizers are not commonly available in research laboratories, whereas the recombinant approach to protein production using prokaryotic expression systems is well established and widely used.

In general, recombinant DNA technology is used to produce peptides with more than 20 amino acids [13], with E. coli being the most popular organism due to its rapid growth, low culture cost, and high productivity [[15], [16], [17], [18]]. However, producing small-sized peptides (<20 aa) in E. coli is difficult because cationic small-sized peptides can be proteolytically degraded and overexpressed peptides can be toxic to the host. Furthermore, some post-translational modifications are difficult to achieve in the E. coli expression system (primarily glycosylation or the formation of disulfide bridges [19]), and the expressed protein tends to form inclusion bodies (due to protein solubility issue). Nonetheless, E. coli SHuffle (New England Biolabs) and Origami (Novagen) have been engineered to promote disulfide bond formation in the cytoplasm. SHuffle strain also lacks the proteases Lon and OmpT, resulting in a decrease in the breakdown of newly synthesized peptides [20,21]. By including an appropriate protein tag, such as thioredoxin A (TrxA), glutathione s-transferase (GST), maltose-binding protein (MBP), and small ubiquitin-related modifier (SUMO), the solubility of recombinant peptides produced in the E. coli expression system can be increased [[22], [23], [24]]. A properly chosen tag can also ensure that the expressed protein has a net negative or neutral charge, avoiding host proteases and preventing potential peptide toxicity [[25], [26], [27]].

Choosing a tag requires accountability of its structure, size, and compatibility with downstream applications. For example, cysteine deficient MBP permits its chemical digestion, however, it is disproportionately larger (42 kDa) than the fused target peptide (2 kDa). Tags can be removed from peptides using proteases such as enterokinase, factor Xa, or SUMO; however, the efficiency of the enzymatic digestion is dependent on surrounding residues or the accessibility of the cleavage site [24,25,28]. Furthermore, an additional step of affinity chromatography is required to eliminate the proteases and the tag after digestion. Chemical fragmentation by volatile reagents such as CNBr (cleavage site Met↓), formic acid (cleavage site Asp↓Pro), and hydroxylamine (cleavage site Asn↓Gly) on the other hand, is a less expensive method and can be used for insoluble proteins as well.

In this paper, we describe in detail the methodology for producing peptides in the E. coli expression system, which allows the formation of disulfide bonds. We also propose a new 6.2 kDa HOE tag that allows for the efficient expression of small peptides while retaining its tertiary structure. Because the tag lacks methionine, it is also suitable for chemical cleavage. To separate the peptide of interest, enzymatic cleavage sites are also included in the HOE tag. The findings of this study validate the experimental approach designed for the production of recombinant small peptides.

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Section snippets

Modification of pQE-30 plasmid

Nucleotide sequence encompassing nts 379 to 462 of OspA of Borrelia garinii (Genbank accession no. GU906888), was amplified using primers ospA F and ospA R (Table 1, ospA R is flanked with factor Xa cleavage site). The amplified sequence is presented in Fig. S1A. Amplicons were digested with BamHI and SalI enzymes (Thermo Fisher Scientific, USA), purified by NucleoSpin kit (Macherey-Nagel, Germany), and ligated into pQE-30 vector (Qiagen, Germany) using T4 DNA ligase (Thermo Fisher Scientific)

The HOE tag

The CX7C DNA template was ligated into the pQE-30-N tag vector to create the new plasmid pQE-30-N tag-II, which allows for the expression of a small-sized peptide fused with the N terminal HOE tag, as shown in Fig. 1. The hydrophilicity prediction of the HOE tag using the PEPTIDE 2.0 server [30] revealed 85.79% hydrophilic amino acid residues, suggesting excellent solubility of the tag. A high percentage of hydrophilic residues can also be found in commonly used tags such as TrxA, SUMO, GST, or

Conclusion

We described a simple procedure for producing small-sized peptides in the E. coli expression system in this study. The method is validated by the production of 21 peptides – both cyclic and linear – fused to a novel HOE tag designed by us, which can be cleaved by either chemical (CNBr) or enzymatic (enterokinase) digestion. The expression cassette could be easily created by inserting the desired nucleotide sequence (encoding the peptide of interest) into an antisense primer composed of constant

Author contributions

Experiments were designed by MB and EM. Primers design and vector modifications were done by MB. Cloning was performed by EM, and PP. Protein constructs were produced by EM, PP, OD and PH. Cyclicity assay was performed by PP. Chemical and enzymatic cleavage was done by EM and PP. EM, AK and MB wrote the manuscript. MB and KB provide funding. All authors read and approved the final manuscript.

Funding

This work was supported by APVV [grant number APVV-18-0259, grant number PP-COVID-20-0044], VEGA [grant numbers 1/0439/18, 1/0105/19, 2/0128/21], and ERA-NET EuroNanoMed III [grant number TJC2018-049]. EM is supported from APVV-18-0259. OD and PP are funded from DSV- ITMS2014+ project code NFP313010V455.

CRediT authorship contribution statement

Evelína Mochnáčová: Methodology, Writing – original draft. Patrícia Petroušková: Methodology. Oľga Danišová: Methodology. Patrícia Hudecová: Methodology. Katarína Bhide: Funding acquisition. Amod Kulkarni: Writing – review & editing. Mangesh Bhide: Conceptualization, Writing – review & editing, Methodology, Funding acquisition.

Declaration of competing interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

NONE.

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