Recombinant Expression and Bioactivity Comparison of Four Typical Fungal Immunomodulatory Proteins from Three Main Ganoderma Species

Background More than a dozen of fungal immunomodulatory proteins (FIPs) have been identified to date, most of which are from Ganoderma species. However, little is known about the similarities and differences between different Ganoderma FIPs’ bioactivities. In the current study, two FIP genes termed FIP-gap1 and FIP-gap2 from G. applanatum, along with LZ-8 and FIP-gsi, another two representative Ganoderma FIP genes from G. lucidum and G. sinense were functionally expressed in Pichia. Subsequently, bioactivities of four recombinant Ganoderma FIPs were demonstrated and compared. Results All the four Ganoderma FIP genes could be effectively expressed in P. pastoris GS115 at expression levels ranging from 197.5 to 264.3 mg L− 1 and simply purified by one step chromatography using HisTrap™ FF prepack columns. Amino acid sequence analysis showed that they all possessed the FIP conserved fragments. The homologies of different Ganoderma FIPs were from 72.6 to 86.4%. In vitro haemagglutination exhibited that FIP-gap1, FIP-gsi and LZ-8 could agglutinate human, sheep and mouse red blood cells but FIP-gap2 agglutinated none. Besides, the immunomodulation activities of these Ganoderma FIPs were as: rFIP-gap2 > rFIP-gap1 > rLZ-8 and rFIP-gsi in terms of proliferation stimulation and cytokine induction on murine splenocytes. Additionally, the cytotoxic activity of different FIPs was: rFIP-gap1 > rLZ-8 > rFIP-gsi > rFIP-gap2, examined by their inhibition of three human carcinomas A549, Hela and MCF-7. Conclusions Taken together, four typical Ganoderma FIP genes could be functionally expressed in P. pastoris, which might supply as feasible efficient resources for further study and application. Both similarities and differences were indeed observed between Ganoderma FIPs in their amino acid sequences and bioactivities. Comprehensively, rFIP-gaps from G. applanatum proved to be more effective in immunomodulation and cytotoxic assays in vitro than rLZ-8 (G. lucidum) and rFIP-gsi (G. sinense). Electronic supplementary material The online version of this article (10.1186/s12896-018-0488-0) contains supplementary material, which is available to authorized users.

It is estimated that there are approximately 200 Ganoderma species around the world, among which more than 80 are identified in China [1][2][3]. Traditional classification of Ganodermas in China was mainly made according to their morphological characteristics [38]. Zhao et al. divided Ganoderma into two main subgenera, Ganoderma and Elfringia. Subsequently, they found that subgenus Ganoderma contained two subsections, subsect. Phaeonema (Zizhi) and subsect. Ganoderma (Lingzhi) [39,40]. Interestingly and coincidentally, FIPs have been identified from G. lucidum (LZ-8 or FIP-glu, Genbank No. M58032.1) and G. sinense (FIP-gsi, Genbank No. AY449805.1), which are two most famous representative species from subsect. Phaeonema and subsect. Ganoderma. Similarly, a pair of novel FIP genes have also cloned from G. applanatum, the typical species of subgenus Elfringia, in our previous study [34,35], which were termed as FIP-gap1 (or known as FIP-gap, Genbank No. JN167598.1) and FIP-gap2 (Genbank No. KX591653). However, very little is known about the exact differences and similarities between different Ganoderma FIPs until now. In the current study, those typical Ganoderma FIP genes from the three representative species were functionally expressed in Pichia pastoris GS115 and purified. Subsequently, a series of functional assays were performed using different recombinant Ganoderma FIPs in order to analyze and to compare their bioactivities comprehensively. Our results indicated that differences indeed existed between different Ganoderma FIPs despite of some similarities in their protein sequences and amino acid compositions. These findings will provide data on the biological functions of different Ganoderma FIPs which will help to elucidate the potential application and development of FIPs in biomedical or therapeutic studies.

Gene synthesis and recombinant vector construction
Four codon-optimized Ganoderma FIP genes were artificially synthesized by Sango (Shanghai, China), within  which a 6 × His tag sequence was also inserted before the stop codon. Codon adaptation indexes (CAI) for Ganoderma FIP genes in P. pastoris before and after codon-optimization are shown in Additional file 2. The sketch map of synthetic FIP genes is as shown in Additional file 3. The eukaryotic expression vectors were constructed via digestion and ligation using synthetic gene fragments and plasmid pPIC9, transformation of E. coli competent cells, and confirmation by colony PCR and sequencing. Finally, recombinant plasmids were obtained and were universally named as pPIC9-His-FIP-Ganoderma (Fig. 3), in which the four Ganoderma FIP genes were integrated under the control of the P AOX promoter.

Expression and purification of Ganoderma FIPs in P. pastoris
After transformation, the correct positive P. pastoris transformants were identified by screening culture on MD (Minimal Dextrose) and colony PCR. The phenotypes of all the yeast transformants were His + Mut s after further cultivation on MD and MM (Minimal Methanol) media. Growth and induction culture of yeast transformants were conducted in YPD and BMMY, respectively. Purification of recombinant Ganoderma FIPs were performed by one-step chromatography using prepacked HIS Trap™ FF columns. The supernatants from induced culture at 96 h and wild-type yeast, along with the purified Ganoderma FIPs, were subjected to SDS-PAGE and Western blot for detection and analyses. Two protein bands of approximately 14 and 17 kDa could be clearly observed for rFIP-gap1 and rFIP-gap2, whereas single protein bands, 14 and 17 kDa, of rLZ-8 and rFIP-gsi were obviously visualized, respectively (Fig. 4a). Moreover, all those protein bands could be obviously immunologically detected by anti-6 × His antibody (Fig.  4b). Besides, the expression level of rFIP-gap1, rFIP-gap2, rLZ-8 and rFIP-gsi were 247.4, 197.5, 253.6 and 264.3 mg L − 1 measured by densitometric scanning and quantitative analysis.

Haemagglutination and mitogenic activities of recombinant Ganoderma FIPs
Hemagglutination activities of recombinant Ganoderma FIPs (all at 5 μg mL − 1 ) towards three different mammalian red blood cells, which was detected using a microscope at magnification of (16 × 20). Results indicated that rFIP-gap1 and rFIP-gsi were able to agglutinate human, sheep and mouse red blood cells, whereas rLZ-8 showed hemagglutination ability to sheep and mouse blood cells, but not the human's. However, the other FIP from G. applanatum, rFIP-gap2, couldn't agglutinate any of the red blood cells (Table 1; Additional file 4). In addition, PHA (5 μg mL − 1 ) used as a positive control caused haemagglutination towards human, sheep and mouse red blood cells. The MTT method was applied to examine the stimulatory activities of recombinant FIPs, which revealed that all Ganoderma FIPs could significantly stimulate the cell viability of murine splenocytes at concentrations of 5 μg mL − 1 by contrast with the negative control (Fig. 5). Interestingly, rFIP-gap1, rFIP-gap2 and rFIP-gsi were even more effective in

Cytotoxic effect assessment of recombinant Ganoderma FIPs
The cytotoxic activities of different Ganoderma FIPs were demonstrated by their inhibitory effects towards three types of human cancer cells using a MTT method as described above. And different IC 50 were calculated from the cell viability data using SPSS 19.0 software. Results indicated that rFIP-gap1 showed significant cytotoxic effect towards A549, Hela and MCF-7 cells, with IC 50 values of 29.89, 8.34 and 12.19 μg/mL, respectively (Fig. 7). rLZ-8 and rFIP-gsi exhibited moderate inhibitory activities to the same cancer cell lines. The IC 50 values of rLZ-8 were 21.65, 17.53 and 43.72 μg/mL, whereas those of rFIP-gsi were 68.04, 19.44 and 30.05 μg/mL (Fig. 7). rFIP-gap2, the other FIP from G. applanatum showed the poorest cytotoxicity. The IC 50 values of rFIP-gap2 towards A549, Hela and MCF-7

Discussion
Ganodermas have been applied and recorded in China for thousands of years, which are deemed as precious auspicious herbs and thought to prolong life as well as to cure the dying patients [1,2]. As estimated, there are over 200 Ganoderma species worldwide with more than 80 distributed in China, among which G. lucidum is the most well-known [3,39,40]. Various components from Ganodermas have been proven bioactive including polysaccharides, proteins, glycopeptides, triterpenes etc. Previous studies found that polysaccharides and triterpenes were the two main active substances for Ganodermas [8,11,40]. In terms of that, Zhao et al. once stated that G. sinense was the most valuable and bio-effective species [40]. However, discovery of FIPs revealed that proteins alone from Ganodermas could exert very similar bioactivities to polysaccharides and triterpenes, such as The relative concentrations were calculated from the reading data at 450 nm by ELISA. All data was statistically analyzed through one-way ANOVA. Different letters indicate significance (P < 0.05) and same letters mean the difference is not significant immunomodulation and anti-tumor activities [23]. Since the first FIP, LZ-8, was identified from G. lucidum, seventeen FIPs have been reported to date, among which more than a half are from Ganodermas [24][25][26][27][28][29][30]. And these Ganoderma FIPs shared very high homology in their amino acid sequences (Fig. 1). On basis of the protein sequence alignment and phylogenetic tree analysis of Ganoderma FIPs (Fig. 2), we could divide Ganodermas into three main groups, Lingzhi (Ganoderma), Zizhi (Phaeonema) and Shushe (G. applanatum) in the current paper. That was very similar and accordant with the traditional classification of Ganodermas as well as other sorting results using molecular markers [41]. Therefore, we can speculate that all Ganodermas probably contain FIPs, which can be potentially used as candidate markers for Ganoderma classification.
As reported previously, it is time-consuming and costly to extract FIPs from natural mycelia and fruiting bodies with usually low yield [25,28]. For example, merely 10 mg of LZ-8 was purified from 300 g wet G. lucidum mycelia [24]. Hence, researchers have been making efforts in investigating new methods to obtain recombinant FIPs via bio-engineering techniques. For instance, FIP-gts and FIP-fve have been expressed in E. coli and insect cells, with yields of ranging from 5 to 20 mg L − 1 [28]. Besides, LZ-8 has been expressed in Bacillus subtilis and Lactococcus lactis at expression levels of 17.4 and 1.24 mg L − 1 , respectively [42]. However, the expression levels of these rFIPs from prokaryotic and insect cells are commonly very low. Furthermore, recombinant FIPs from E. coli possessed only 50% bioactivity of native ones [42]. The P. pastoris expression system is regarded as an ideal host and extensively employed to express functional proteins of medicinal or commercial values [28,31]. Generally, the expression levels are high as a result of the P AOX promoter, which is reported to be the strongest promoter to date, and is regulated by methanol [43]. Besides, recombinant heterogeneous proteins can be produced extracellularly and secreted into the culture media with the help of α-signal peptide, which effectively facilitates the downstream processing and purification. In addition, cultivation and fermentation of P. pastoris is simple and economical, making it quite applicable for scale-up fermentation and even industrial production [43,44]. Given all these advantages, P. pastoris GS115 was adopted as a host to express the four typical Ganoderma FIP genes in the current study. Results exhibited that they were all effectively expressed after codon-optimization, with expression levels of 197.5-264.3 mg L − 1 (Fig. 4), which is accordant with previous reports (Table 2) [45]. Additionally, two protein bands of rFIP-gap1 and rFIP-gap2 could be clearly observed in SDSPAGE and Western blotting (Fig. 4). The reason for that was probably due to the post-transcriptional modification including glycosylation, which was a common phenomenon in P. pastoris expression system [31,43]. Potential glycosylation sites were found therein the FIP-gaps' amino acid sequences, including 38 Asn- 39 Leu- 40 Thr in FIP-gap1 and 31 Asn-32-Pro- 33 Ser in FIP-gap2. Fig. 7 Cytotoxic effects of four Ganoderma FIPs towards A549 (a), Hela (b) and MCF-7 (c) cancer cell lines. All cells were treated with rFIP-gap1, rFIP-gap2, rLZ-8 and rFIP-gsi at concentration ranging from 2 and 32 μg mL − 1 . The inhibitory rates were calculated according to cell viabilities measured by MTT assays and the half-maximal inhibitory concentration (IC 50 ) was deduced from the graphs A series of biological activities have been reported since the discovery of FIPs, including haemagglutination, anti-allergy, immunomodulation, anti-tumor and anti-virus [23][24][25]28]. Nevertheless, quite little is known about the similarities and differences between the Ganoderma FIPs. Thus, bioactivities were examined and compared specifically between FIP-gap1, FIP-gap2, LZ-8 and FIP-gsi, the four rFIPs from three representative Ganodermas, in terms of haemagglutination, mitogenesis, cytokine induction and cytotoxic effect. Primarily, rFIP-gap1 and rFIP-gsi showed strong haemagglutination ability, as they could agglutinate human, sheep and mouse red blood cells (Table 1). And rLZ-8 was capable to agglutinate sheep and mouse red blood cells but not the human's, which is identical to natural LZ-8 and rLZ-8 s from E. coli or P. pastoris [44]. However, rFIP-gap2, the second FIP from G. applanatum, could agglutinate none of the three blood cells (Table 1). Later, the immunomodulation activities of these Ganoderma FIPs were evaluated by their mitogenesis and cytokine induction towards murine spleen lymphocytes. Results indicated that rFIP-gap2 had the strongest stimulation to enhance cell viability of mouse splenocytes, whereas rFIP-gap1 and rFIP-gsi showed moderate mitogenesis ability, and rLZ-8 was less effective in mitogenic activity (Fig. 5). For cytokine induction test, the ability of Ganoderma FIPs to increase IL-2 release were as follows: rFIP-gap1 > rLZ-8 > rFIP-gap2 > rFIP-gsi (Fig. 6a), while the activity of different FIPs to induce IFN-γ expression were: rFIP-gap2 > rFIP-gsi > rFIP-gap1 > rLZ-8 (Fig. 6b). Finally, the cytotoxic effects of different Ganoderma FIPs were detected by their inhibition towards different human cancer cells via a MTT method. Results revealed that four Ganoderma FIPs showed better cytotoxic activity towards Hela and MCF-7 cancer cells than lung cancer A549. To be more specific, the inhibitory ability of different Ganoderma FIPs were: rFIP-gap1 > rLZ-8 > rFIP-gsi > rFIP-gap2, estimated by their IC 50 towards different cancer cell lines (Fig. 7). Additionally, Ganoderma FIPs showed similar but weaker cytotoxic activities by contrast with some other FIPs, for example, FIP-lrh from Lignosus rhinocerotis, exhibited stronger cytotoxicity to A549, Hela and MCF-7 cancer cells, with IC 50 values ranging from 5.07 to 8.94 μg/mL [46]. And the IC 50 value of another FIP, FIP-sch3 (Stachybotrys chartarum), was 10.80 μg/mL [29]. Besides, the cytotoxicity of rFIP-gaps towards A549 and Hela cells in current study was accordant with those in previous reports with very similar IC 50 values [35].

Conclusion
In the current study, a pair of novel FIP genes from G. applanatum, along with another two FIP genes from G. lucidum and G. sinense, were functionally expressed in P. pastoris GS115, at yields ranging from 197.5 to 264.3 mg L − 1 , which were with much higher yield than recombinant FIPs from E. coli and insect cells. Bioactivity examination of these four typical Ganoderma FIPs indicated that FIP-gap1 and FIP-gap2 showed more immunomodulation activity than LZ-8 and FIP-gsi in terms of mitogenesis and cytokine induction from mouse splenocytes. Besides, cytotoxic effects of these FIPs were: rFIP-gap1 > rLZ-8 > rFIP-gsi > rFIP-gap2 towards three human cancer cell lines after comparison of their IC 50 . Our results showed that similarities and differences indeed existed between different Ganoderma FIPs in both their protein sequences and bioactivities. And it seemed that FIPs from G. applanatum were more bioactive than FIPs from G. lucidum and G. sinense. Additionally, our study also confirmed that recombinant FIPs from P. pastoris could be used as robust effective resources for further development and investigation.

Materials and methods
Strains, plasmids and kits P. pastoris strain GS115 and plasmid pPIC9 were purchased from Invitrogen (USA, Catalog number: K171001). Escherichia coli Top10 competent cells were procured from Tiangen (Beijing, China). HisTrap™ FF prepack columns (5 mL) were GE products (USA). Frozen-EZ Yeast Transformation II Kit™ was from Zymo Reasearch (USA). Mouse IL-2 and IFN-gamma ELISA kits were bought from Beyotime (Shanghai, China). Restriction endonucleases, T4 DNA ligase and agarose gel DNA purification kits were Takara (Japan) products. Concanavalin A (ConA) and biotin were from Sigma (USA). Thiazolyl blue and yeast nitrogen base with ammonium sulfate without amino acids (YNB) were purchased from Genview (USA). All the other chemicals were of analytical grade. Gene synthesis and sequencing were carried out by Sangon (Shanghai, China).

Sequence alignment and phylogenetic tree construction
Up till now, ten Ganoderma FIPs have been identified as mentioned above. Primarily, nucleotide and protein sequcences' BLAST searching and analysing was carried out using NCBI and DNAMAN software (version 8.0). Then the phylogenetic tree of Ganoderma FIPs was constructed using the neighbour-joining method by the MEGA in version 7.0 software (http//https://www.megasoftware.net). The statistical confidence in the phylogenetic relationships was assessed with bootstrap tests, which were replicated 1000 times.

Gene synthesis and vector construction
Four typical FIPs from three famous representative Ganoderma species were chosen for targets including LZ-8, FIP-gsi, FIP-gap1 and FIP-gap2. Firstly, the low-usage and rare codons in the original four FIP genes were replaced by high-usage codons, according to the codon bias in P. pastoris [47]. Secondly, EcoRI and NotI recognition sequences were added before the start codons and after the stop codons, respectively. In addition, 6 × His-tag sequences were also inserted before stop codons in the C terminal. Then the four optimized Ganoderma FIP genes were sent to Sangon for synthesis. Both the synthetic FIP genes and plasmid pPIC9 were digested with EcoRI and NotI at 37°C for 1.5 h. Then a rapid ligation using target genes and digested pPIC9 was conducted at 22°C for a half hour. The ligated product was used to transform E. coli TOP10 competent cells, which were later screened on LB media with ampicillin (100 μg mL − 1 ). The positive E. coli transformants were detected by colony PCR and endonuclease digestion. Finally, the correct recombinant plasmids were obtained and designated as pPIC9-His-FIP-Ganoderma, which were further confirmed by sequencing.

Transformation and induced expression of Ganoderma FIPs
Preparation and transformation of yeast competent cells were performed according to the kit instruction (Catalog number: T2001). Phenotype of yeast transformants was determined by screening on MD and MM media. Positive transformants were verified by colony PCR and cultured in YPD at 30°C, 200 rpm until OD 600 ≥ 2.0. Then the yeast cells were harvested by centrifugation at 6000 rpm for 5 min and then resuspended in BMMY 200 mL 500 mL − 1 in one flask) to induce protein expression. Methanol was added every 24 h to maintain a final concentration of 1% (v/v). At 96 h, 1 mL of the expression cultures were transferred for later use. The wild-type yeast was used as a negative control and cultured in a same way. Purification of recombinant Ganoderma FIPs was carried out using HisTrap™ FF prepack columns according to the manufacturer's protocol (Catalog number: 17-5319-01). The protein masses of recombinant proteins were determined through SDS-PAGE and quantitative analyses with an image-analyzing system (QUANTITY ONE, Bio-Rad, USA) [28].

SDS-PAGE and Western blot analyses
25 μL supernatants of four recombinant Ganoderma FIPs (at induction time of 96 h) and 10 μL purified protein samples were mixed with equal volumes of 2 × loading buffer for SDS-PAGE test. The wild-type yeast supernatant was as a negative control. All the mixtures were boiled for 5 min before electrophoresis. SDS-PAGE was performed in 12% acrylamide gels and visualized by staining with Coomassie brilliant blue R250. For Western blot, after SDS-PAGE, proteins samples were transferred onto a PVDF membrane. After blocking with 5% nonfat dry milk in TBST and incubated with anti-His monoclonal antibody raised in mouse (Tiangen, China, 1:25000 diluted) for two hours. The membrane was washed five times with TBST and incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG raised in goat (Tiangen, 1:5000 diluted) for 1.5 h at 37°C [43].After incubation with the ECL reagent (Pierce, USA), the membrane was subjected to X-ray film for exposure 2-5 s at totally dark conditions. Subsequently, the X-ray film was developed in the developer bath for 15-20 s, and then fixed in the fixer solution.

Haemagglutination and mitogenesis assays of recombinant FIPs
After purification, the agglutination activity of recombinant Ganoderma FIPs was primarily tested. 50 μL of purified rFIP-gap1, rFIP-gap2, rLZ-8 and rFIP-gsi (final concentration of 5 μg mL − 1 ) in PBS were mixed with equal volumes of human, sheep and mouse red blood cells (1.5%, v/v) in a 96-well microplate, respectively. PHA (5 μg mL − 1 ) served as a positive control, whereas PBS was used as a negative control. The plate was incubated in a 5% CO 2 incubator at 37°C after gently mixed and haemagglutination was examined after 1.5 and 24 h. The mitogenic activity of Ganoderma FIPs was demonstrated by their stimulatory effect towards mouse splenocytes. Homogenous splenocytes of male Balb/c mice (4-6 weeks old, purchased from the Pharmacology Experimental Center of Shenyang Agriculture University) were grown and suspended to 1 × 10 6 cells mL − 1 in RPMI 1640 medium. Subsequently, 100 μL cell suspension and recombinant Ganoderma FIPs (5 μg mL − 1 ) were seeded into a 96-well plate. Concanavalin A (ConA, 5 μg mL − 1 ) and PBS served as positive and negative controls. The cell viability was evaluated by a MTT method. In brief, after the cells were cultured in 5% CO 2

Cytokines induction detection
To test the immunomodulatory bioactivities of recombinant Ganoderma FIPs, the expression levels of induced IL-2 and IFN-γ released from the murine splenocytes were detected by ELISA method. The murine splenocytes were prepared as above and adjusted to 1 × 10 7 cells mL − 1 in RPMI 1640 medium. Then 100 μL cells plus equal volumes of rFIP-gap1, rFIP-gap2, rLZ-8 and rFIP-gsi (final concentration of 5 μg mL − 1 ) were mixed and seeded into a 96-well plate. Meanwhile, PBS and ConA (5 μg mL − 1 ) were used as controls. After the plate was cultured in 5% CO 2 at 37°C for 48 h, the supernatants of the cells were collected. Expression levels of mouse IL-2 and IFN-γ in the supernatants were measured using ELISA kits (Multi Sciences, Hangzhou, China) in accordance with the manufacturer's instructions, and serial concentrations (7.81, 15.63, 31.2, 62.5, 125 and 250 pg mL − 1 ) and (62.50, 125, 250, 500, 1000 and 200 pg mL − 1 ) of mouse IL-2 and IFN-γ were prepared as standards, respectively [28,43].

Cytotoxicity examination of recombinant Ganoderma FIPs
To determine cytotoxic activities, three cancer cell lines, human lung carcinoma A549, cervical carcinoma Hela and human breast carcinoma MCF-7 were used for test, which were provided by the Cell Research Center, College of Veterinary Science and Animal Husbandry (SYAU). Those cancer cells were cultured in DMEM (Tiangen) supplemented with 10% FBS, 100 units/mL penicillin and 100 μg mL − 1 streptomycin at 37°C in a 5% CO 2 incubator. Subsequently, the cells were trypsinized with 0.25% trypsin containing 0.04% EDTA and adjusted to 5 × 10 5 cells mL − 1 . 100 μL cancer cells were seeded in triplicate wells with diluted rFIP-gap1, rFIP-gap2, rLZ-8 and rFIP-gsi (final concentration of 2, 4, 8, 16 and 32 μg mL − 1 ), which were continually cultured for another 24 h. Meanwhile, PBS was added into these cell cultures in a same way as a negative control. Inhibition of cancer cell growth was presented using the half-maximal inhibitory concentration (IC 50 ), which was defined as the concentration causing 50% inhibition of cell proliferation [28].

Data analysis
Data were presented as the mean ± SD of three separate experiments performed in duplicate. Statistical analysis was performed through means of one-way ANOVA and the LSD-test with SPSS 19.0 software. Differences were considered to be statistically significant when the P < 0.05.