Optimized expression of Peptidyl-prolyl cis/transisomerase cyclophilinB with prokaryotic toxicity from Sporothrix globosa

Abstract   Cyclophilin B (CypB), a significant member of immunophilins family with peptidyl-prolyl cis-trans isomerase (PPIase) activity, is crucial for the growth and metabolism of prokaryotes and eukaryotes. Sporothrix globosa (S. globosa), a principal pathogen in the Sporothrix complex, causes sporotrichosis. Transcriptomic analysis identified the cypB gene as highly expressed in S. globosa. Our previous study demonstrated that the recombinant Escherichia coli strain containing SgcypB gene failed to produce sufficient product when it was induced to express the protein, implying the potential toxicity of recombinant protein to the bacterial host. Bioinformatics analysis revealed that SgCypB contains transmembrane peptides within the 52 amino acid residues at the N-terminus and 21 amino acids near the C-terminus, and 18 amino acid residues within the cytoplasm. AlphaFold2 predicted a SgCypB 3D structure in which there is an independent PPIase domain consisting of a spherical extracellular part. Hence, we chose to express the extracellular domain to yield high-level recombinant protein with PPIase activity. Finally, we successfully produced high-yield, truncated recombinant CypB protein from S. globosa (SgtrCypB) that retained characteristic PPIase activity without host bacterium toxicity. This study presents an alternative expression strategy for proteins toxic to prokaryotes, such as SgCypB. One-Sentence Summary The recombinant cyclophilin B protein of Sporothrix globosa was expressed successfully by retaining extracellular domain with peptidyl-prolyl cis-trans isomerase activity to avoid toxicity to the host bacterium.


Introduction
Cyclophilin B (CypB) belongs to the immunophilins family and has peptidyl-prolyl cis-trans isomerase (PPIase) activity, which catalyzes the cis-trans isomerization process of proline residues, widely existing in eukaryotes and prokaryotes (Wang & Heitman, 2005 ).Cyclophilins (Cyps) are intracellular protein targets of the immunosuppressive drug cyclosporin A (CsA), and the PPIase activity is strongly inhibited by CsA through binding PPIase active site (Swanson et al., 1992 ;Walsh et al., 1992 ). Fungal PPIases are widely involved in a range of biological processes, including virulence, growth, stress response, and metabolic regulation (Dimou et al., 2017 ;Singh et al., 2018 ).Studies on Cryptococcus neoformans showed that Cyps are pivotal in growth, mating, and virulence.Their absence or mutation diminishes the stress tolerance of fungi.(Wang et al., 2001 ).Infection experiments with Beauveria bassiana on Plutella xylostella larvae indicated that CypB upregulation is associated with fungal invasion and virulence (Collette & Lorenz, 2011 ).Given the conserved nature of Cyps across fungi, they may play analogous roles in pathogenesis, such as adhesion and invasion.(Chen et al., 2011 ).Recently, a nanoscale liquid chromatography coupled with tandem mass spectrometry approach was used in a research to provide yeast proteomic profiles of Sporothrix .Cyclophilin B was identified, showing a higher expression in Sporothrix globosa ( S. globosa ) .(Silva-Bailão et al., 2021 ) .
Sporothrix is an etiological agent of sporotrichosis, which is an implantation mycosis, mainly including S. schenckii , S. brasiliensis , and S.globosa.The most common clinical manifestations of this mycosis are acute or chronic cutaneous and lymphocutaneous lesions, occurring after contact with soil, sphagnum moss, thorny plants, and even certain animals like cats, preferably in tropical and subtropical regions (Chakrabarti et al., 2015 ;Nava-Pérez et al., 2022 ).Research on Sporothrix virulence has predominantly focused on S. schenckii and S. brasiliensis , noted for higher virulence than S. globosa .(Tamez-Castrellón et al., 2020 ).However, virulence factors associated with S. globosa were poorly described, among which more were melanin and the fungal cell wall components, such as polysaccharide, chitin, Gp70, Gp60, and enolase (Ruiz-Baca et al., 2009 ;Alba-Fierro et al., 2016 ;Félix-Contreras et al., 2020, Portuondo et al., 2019 ;Villalobos-Duno et al., 2021 ).Our previous bioinformatical analysis found CypB was a potential membrane protein, located in the surface of S. globosa (Zixian et al., 2023 ) .However, we were unable to obtain the full-length recombinant protein as the bacterium lysed upon induction.Hence, it was necessary to optimize the expression of Sg CypB.
Previous studies have shown that the prokaryotic expression of eukaryotic membrane proteins is a great challenge, leading to low yield and easy formation of inclusion bodies (He et al., 2014 ).The main reason is the membrane proteins are hydrophobic, resulting in a toxic effect on host cells (Kesidis et al., 2020 ).At the same time, a large amount of recombinant eukaryotic membrane proteins expressed on the host membrane can change its characteristics and cause toxicity to the host cells.Therefore, prokaryotic expression of membrane proteins mainly expresses the soluble region, not the full-length protein (Laage & Langosch, 2001 ).This is because of the differences between prokaryotic and eukaryotic membranes and the lack of cellular environment such as posttranslational modification during expression (Bill et al., 2011 ).Despite these limitations, bacterial cells remain a popular choice for their cost-effectiveness, high yield potential, and the relative ease of genetic manipulation.(Kesidis et al., 2020 ).Meanwhile, to the best of our knowledge, little is known about the prokaryotic expression for full length of eukaryotic CypB protein, and that only a hydrophobic leader sequence was truncated in the prokaryotic expression of human CypB has been reported (Price et al., 1991 ).Consequently, we only retained the extracellular part of Sg CypB, with the aim of obtaining a large amount of recombinant protein with PPIase activity, as it is involved in the pathogenesis of sporotrichosis.
We are reporting the successful high-level expression of Sg CypB protein with the PPIase enzymatic activity in Escherichia coli (E.coli) BL21(DE3) cells using the pET30a( + ) expression vector system.This approach represents a viable option for expressing proteins that are toxic to prokaryotic systems.

Source of S. Globosa cypB Gene Sequence
The whole genome sequence of S. globosa (ASM163044v1, GenBank assembly accession: GCA_001 630 445.1) was gained by retrieving the National Center for Biotechnology Information (NCBI).The CDS sequence of SgcypB gene was obtained via sequence alignment with the CDS sequence of the S. schenckii cyclophilin B gene ( SscypB ) (NCBI Reference sequence: XM_016 733 058.1) and the whole genome sequence of S. globosa .

Structure and Function Analysis By Bioinformatics
DeepTMHMM ( https://dtu.biolib.com/DeepTMHMM) was utilized to predict the transmembrane regions of the putative fulllength Sg CypB.AlphaFold2 (v2.0) ( https://github.com/deepmind/alphafold) were employed to predict the structure.The highest ranking structural model of Sg CypB and Sg trCypB were selected as the receptors for molecular docking.The structure of CsA was the ligand for molecular docking, downloaded directly from Pub-Chem ( https://pubchem.ncbi)(PubChem ID:5 284 373).The potential natural binding sites were predicted using Discovery Studio 2019.LibDock pattern was used for high-precision docking.Entrusting receptors and ligands with CHARMm forcefields to estimate the binding free energy.GROMACS 2023 was used to calculate the average root mean square deviation (RMSD) and radius of gyration (Rg) of each docking result.The open-source version of PyMol ( https://sourceforge.net/projects/pymol/) was applied to prepare the images.

The Synthesis of the Gene Encoding the Extracellular Part of Sg CypB Protein and the Construction of the Recombinant Plasmid
Through our previous analysis of the characterization of Sg CypB, according to the amino acid sequence of the extracellular part, we chose the codons preferred by E.coli to artificially synthesize the DNA fragment Sg trCypB encoding 218 amino acid residues.The DNA fragment was synthesized and cloned into pET30a( + ) vector through Nde I and Xho I sites by Tsingke (Guangzhou, China).The His-tag at C-terminal of this vector was retained for purification and identification.The recombinant expression vector pET30a-Sg trCypB was transformed into host bacterium E. coli strain BL21(DE3).

Prokaryotic Expression of Sg trCypB
The recombinant E. coli BL21(DE3) was grown in Luria-Bertani (LB) broth with kanamycin until the OD 600 reached 0.5.Subsequently, 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to induce expression of the Sg trCypB.The mixture was incubated in a thermostatic shaker at 200 rpm and 37°C for 2 hr, 4 hr, and 6 hr, then 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were performed to identify the prokaryotic expression preliminarily and determine the optimum induction time.The number of viable bacterium carrying Sg CypB or Sg trCypB was assessed by determining the number of colonyforming units (CFUs) from the LB broth of different induction time, which was done by plating an appropriate dilution of the culture medium on LB agar plates.Meanwhile, the OD 600 of different induction time was determined by spectrophotometer.

Purification of Recombinant Sg trCypB
We then chose the optimum induction time to induce expression of the Sg trCypB in the 1 L of fresh LB medium containing 50 μg/mL kanamycin.The bacteria were then harvested and disrupted by sonication.The cells were lysed and then centrifuged to separate the supernatant and pellet.The SDS-PAGE method was used to identify the location of the recombinant protein.The supernatant filtered through a 0.45 μm pore membrane, and the Ni NTA Beads, which was purchased from Changzhou Smart-Lifesciences Biotechnology Co., Ltd (Changzhou, China), was utilized to purify the fusion protein in the supernatant according to the operating instructions.The recombinant proteins were dialyzed in phosphate-buffered saline for 18 hr at 4°C.Protein concentrations were measured using BCA assay method in accordance with the manufacturer's instructions.

Western Blot Analysis
To identify the Sg trCypB, 12.5% SDS-PAGE and western blot analysis were performed.Protein samples were transferred to a polyvinylidene difluoride membrane (Merck, Germany) via electroblotting.The membrane was then blocked with 5%(w/v) skimmed milk powder in TBST (Tris Buffered Saline with Tween) for 1 hr and subsequently incubated with 1:1000 dilution rabbit anti-His antibody at 4°C overnight.After being washed with TBST three times, the membrane was incubated with 1:5000 dilution HRP-conjugated goat anti-rabbit IgG antibody at room temperature for 1 hr.

PPIase Activity Assay
Peptidyl-prolyl cis-trans isomerase activity of the Sg trCypB was determined with an α-chymotrypsin-coupled PPIase assay (Fischer et al., 1992 ).A total mixture of 1 mL (100 μM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, 35 nM HEPES, 10 μM α-chymotrypsin, and Sg trCypB) was utilized, while double steam water was used to take the place of the Sg trCypB as the negative control.The reaction system (without α-chymotrypsin) was pre-cooled to 15°C.After adding α-chymotrypsin to the system, we used a spectrophotometer to determine the enzyme activity by continuously monitoring the change in absorbance at 390 nm.

Statistical Analyses
The experiments were repeated at least three times.Data were presented as means ± standard deviations (mean ± SD) and analyzed using unpaired t -test or Mann-Whitney U test with GraphPad Prism 8.4.0 and SPSS 21.0 software.A p -value less than 0.05 was regarded as statistically significant.

The Topological Structure of Sg CypB and Optimized Encoding Sequence
The gene sequence was 930 bp long and encoded a putative protein containing 309 amino acids.( Supplementary Fig. S1).A 52amino acid and 21-amino acid transmembrane regions located in 1-52aa and 271-291aa, respectively, predicted by DeepTMHMM (Fig. 1 ).Cyclophilin-type PPIase domain located in 63-220aa, with a signature pattern in 104-121aa, so the extracellular segment included all important binding and catalytic sites of PPIase.Then, to reduce the length and complexity of encoding sequence, we chose to truncate the transmembrane as well as intracellular regions and retain the extracellular domain, finally the sequence of Sg trCypB was determined, and the 3D structure of Sg CypB and Sg trCypB were illustrated in Fig. 1 b.The transmembrane segments consisted of two α-helices located in the N and C terminal.The extracellular domain consisted of seven segments of βlamination and two segments of α-helix forming a classical stable barrel structure with an inner hydrophobic and outer hydrophilic structure ( Supplementary Fig. S2).

Docking of CsA to Sg trCypB Compared With Sg CypB
Discovery Studio 2019 predicted that Sg trCypB and Sg CypB contain six and seven potential active pockets, respectively, but only the apical active pocket of the extracellular domain can stably docking with CsA, which is a natural CsA binding site based on previous amino acid sequence analysis (Zixian et al., 2023 ).Of the 50 docking results generated for each protein, the docking result with the highest score (LibDock score) was selected to display 2D and 3D interactions.Including hydrogen bond donor/acceptor and aromatic edge/face, the results were shown in Supplementary Fig. S3.The interactions between CsA and Sg trCypB or Sg CypB were dominated by the hydrophobic interactions between alkyl groups.Cyclosporin A also formed two conventional hydrogen bonds with Arg and Ala, and two hydrocarbon bonds with Pro and Thr.In order to evaluate the stability, complexes with binding energy less than 0 kcal/mol in LibDock docking results were selected to calculate the average RMSD and Rg values.

The Comparison of Prokaryotic Expression Between Sg CypB and Sg trCypB in Different Induction Time By SDS-PAGE
To preliminarily identify the expression of the recombinant protein, we performed SDS-PAGE with bacteria pellet at different induction times (Fig. 3 ).The result showed that the yield of Sg CypB was lower than Sg trCypB.Meanwhile, we found that the culture medium became clear and the number of host bacteria was less when the Sg CypB protein was expressed ( p < 0.001), while the growth of host cells was unaffected when Sg trCypB protein was expressed ( p > 0.05).(Fig. 4 ).These indicated that prokaryotic expression of full-length membrane protein Sg CypB produced toxicity to the host bacteria, resulting in the low yields of recombinant protein, which could be avoided by removing the trans-membrane domains.

Expression, Purification, and Identification of Sg trCypB and the PPIase Assay of Recombinant Sg trCypB Using Double Steam Water As Negative control.
The Sg trCypB was expressed as a fusion protein with a His-tag after being induced by IPTG.The recombinant protein was mainly To verify whether the Sg trCypB protein retained PPIase activity, the purified Sg trCypB was tested with an α-chymotrypsin-coupled PPIase assay.As a result, the data demonstrated that there existed cis-to -trans conversion in the group of Sg trCypB protein compared with the negative control of double steam water, representing the enzymatic activity (Fig. 6 ).

Discussion
In this study, we report a strategy to achieve high-level expression of Sg trCypB in E. coli cells, only retaining the vital extracelluar domain, thus reducing the size and structural complexity of recombinant protein while preserving enzyme activity.Homology modeling and molecular docking results predicted that Sg trCypB retained the active site in its 3D structure and that CsA could still bind to it.Additionally, we successfully obtained soluble and highlevel Sg trCypB proteins that were identified specifically by anti-His antibody and the recombinant Sg trCypB maintained PPIase activity proved by PPIase assays.
We attempted to express the entire length of Sg CypB in E. coli BL21(DE3) cells.However, the quantity of recombinant protein obtained was insufficient and impure to conduct further research, despite using a gene sequence that was synthesized with E. coli preferred codons.During the process of induction, we observed that the culture medium became clear and the number of colonies decreased.This may be due to the death of host cells, which is consistent with other research on the prokaryotic expression of eukaryotic membrane protein (Grisshammer, 2006 ;Wagner et al., 2007 ).These studies suggest that producing membrane proteins in the cytoplasmic membrane may be toxic to E. coli .This is likely because a large amount of eukaryotic membrane protein needs to be expressed and inserted into the membrane of host cell, which can alter the metabolism or cytomembrane structure of E. coli .However, as an important member of immunophilin family, CypB plays a role of molecular chaperon and may not disturb biological processes in the host bacterium, so it may destroy the cytoplasm membrane and cell wall.
Human CypB prokaryotic expression studies have excluded the initial 25 amino acids due to a non-conserved hydrophobic leader sequence (Price et al., 1991 ).The process of fully expressing eukaryotic CypB in E. coli posed a significant challenge.In the specific case of Sg CypB, the transmembrane regions located in 1-52aa and 271-291aa were also hydrophobic, leading to a low yield and impure protein from E. coli .Little hydrophobic amino acid identified with tandem mass spectrometry (LC-MS/MS) in the recombinant Sg CypB protein proved the prokaryotic expression was a difficult task (Zixian et al., 2023 ).Optimizing expression in E. coli BL21(DE3) cells was necessary to obtain more proteins due to its cost-effectiveness, convenience, ease of operation, and time-saving benefits for prokaryotic expression.For the purpose of studying the enzymatic activity and function of the extracellular domain, we removed the sequence of transmembrane regions, including the following intracellular peptide which were not necessary.We predicted that the key residues in the Sg CypB protein were consistent with the Sg trCypB protein.This suggests that Sg trCypB protein is capable of binding to CsA and retaining its PPIase activity.As a result, the smaller size and simpler protein structure of Sg CypB protein had permitted its effective expression in prokaryotic expression systems, and Sg trCypB protein with PPIase activity was expressed successfully at a high level in E. coli cells.
It has been reported that human cyclooxygenase-2 (COX-2) protein was truncated to be expressed in E. coli BL21(DE3) cells.The truncated protein (trCOX-2) also maintained its enzyme activity.This study demonstrates the feasibility of expressing only the functional domain of a eukaryotic membrane protein in a prokaryotic expression system (Liao et al., 2017 ).Another approach was to express the extracellular domain of human programmed cell death 1 ligand 1 (PD-L1) in prokaryotes and investigate its function in order to identify potential drugs or antibodies.(Kalim et al., 2017 ).However, the human trCOX-2 protein and PD-L1 extracellular domain were mostly expressed as inclusion bodies, different from the Sg trCypB protein in our study, retaining natural conformation and biological function, which might result from the different molecular structure.In this study, PPIase activity of the recombinant protein was tested upon Sue-Ala-Xaa-Pro-Phe-4-nitroanilide, which is the most commonly used substrate of isomer-specific proteolysis using chymotrypsin as the protease.As shown in previous study, the PPIases of Cyps have been studied using tri-and tetrapeptide substrates and the refolding of denatured proteins.Chymotrypsin shows specificity for the trans-A-P amide conformer which it cleaves to produce the chromophore p-nitroaniline in a coupled assay, reflecting the PPIase activity (Fischer et al., 1992 ;Walsh et al., 1992 ).
However, we did not achieve high-level and pure yields of fulllength Sg CypB.Future studies may explore yeast expression systems for full-length protein conformation and functional studies.This research did not compare PPIases between Sg trCypB and other Cyps, a topic for future research, focusing instead on assessing the enzymatic activity of optimized protein.
In conclusion, our approach provides a method to produce substantial quantities of functional Sg trCypB via prokaryotic expression, useful for studies requiring only the functional domain.Furthermore, Sg trCypB could be instrumental in developing CypB inhibitors.This strategy offers a valuable blueprint for expressing CypB proteins from eukaryotic organisms in prokaryotic systems and a method to optimize prokaryotic expression of eukaryotic membrane proteins, minimizing cytotoxicity, and achieving overexpression in E. coli .

Fig. 1 .
Fig. 1.Prediction of transmembrane region of Sg CypB and 3D structure of Sg CypB and Sg trCypB.(a) From prediction of transmembrane region, retaining the sequence of outside was the Sg trCypB.(b) In the 3D structure of Sg CypB, the dashed area was the Sg trCypB.