Science International  Volume 1 Issue 6, 2013

Research Article

Cloning and Overexpression of a DhIFN-α1 Gene from the Halophilic Yeast of Debaryomyces hansenii into Pichia methanolica to Enhance its Tolerance to Salt and Temperature Stresses
Hsiu-Fung Chao
Tainan District Agricultural Research and Extension Station, Council of Agricultural, Executive Yuan, 624, Taiwan

Rongrong Prasenmool
Department of Bioagricultural Science, National Chiayi University, Chiayi, 600, Taiwan

Yung-Fu Yen
Department of Bioagricultural Science, National Chiayi University, Chiayi, 600, Taiwan

ABSTRACT:
The interferon-α1 (IFN-α1) gene, a gene of type I interferons (IFNs), was cloned from Debaryomyces hansenii under 2.5 M of salt stress. DhIFN-α1 was constructed into a pMETB vector that carries the promoter PAUG1, which was induced by methanol. The expression cassette of PAUG1/DhIFN-α1/V5/His was excised from the vector and transformed Pichia methanolica through electroporation. The gene and expression of the transformant DhIFN-α1 was detected by PCR and dot blotting, respectively. The enhanced tolerance to salt and temperature stress of the transformant was verified by exposing it to 1.5 M of salt stress and a low and high temperature of 5 and 37C, respectively. DhIFN-α1 has been successfully cloned and expressed in P. methanolica. This study confirmed that DhIFN-α1 might enhance the tolerance of organisms to salt and temperature stress.
 
    How to Cite:
Hsiu-Fung Chao, Rongrong Prasenmool and Yung-Fu Yen , 2013. Cloning and Overexpression of a DhIFN-α1 Gene from the Halophilic Yeast of Debaryomyces hansenii into Pichia methanolica to Enhance its Tolerance to Salt and Temperature Stresses. Science International, 1: 222-229
DOI: 10.17311/sciintl.2013.222.229
 


INTRODUCTION
Debaryomyces hansenii is one of the most salt tolerant species of yeast and is usually found in salty environments such as the sea and in salty foods1. Kushner2 categorized halophiles as slight halophiles (with optimal growth at 3% NaCl), moderate halophiles (optimal growth at 3%-15% NaCl) and extreme halophiles (optimal growth at 25% NaCl). Sodium chloride significantly improved the growth of D. hansenii, which could tolerate a salinity level of up to 24% (4.11 m) NaCl3 and was designed to be a halophilic yeast4. Methylotrophic yeasts are used to express foreign proteins, especially P. methanolica possesses 2 genes (MOD1 and MOD2), which are induced by methanol5; however, it is a salt-sensitive yeast compared to D. hansenii6 .

Interferon-α1 (IFN-α1) is a gene of type I interferons (IFNs), which are a group of cytokines possessing various biological functions including inhibition of proliferation, induction of differentiation, modulation of the immune system and inhibition of angiogenesis7,8 . IFNs function primarily as a first line of defense against viral infection by inducing the expression of genes that interfere with viral replication and act as natural killer cells9. IFNs are usually used in the medical treatment of various illnesses including tumors, viral infections, autoimmune disorders and neurological syndromes10. However, IFNs also appear to play an integral role in several autoimmune diseases11. Kubo et al.12 reported that IFN-α, β, or γ combinations result in synergistic cell suppression.

In an experiment, Chao et al.6 inoculated D. hansenii under salt stress and then cloned the DhIFN-α1 gene from D. hansenii using cDNA forward subtractive hybridization. P. methanolica was transformed with a DhIFN-α1 gene to examine how DhIFN-α1 demonstrated stress tolerance. These results suggest that the stress tolerance of transformant P. methanolica was enhanced by the overexpression of the DhIFN-α1 gene.


MATERIALS AND METHODS
Yeast strains and inoculations: The halophilic yeast species, Debaryomyces hansenii strain BCRC No. 21947, isolated from Hsilo County, Taiwan, was obtained from the BCRC, Food Industry Research and Development Institute, Taiwan. The Pichia methanolica strain PMAD16 genotype ade-16 was obtained from (Invitrogen, USA). D. hansenii was inoculated at 24°C in a YM11 liquid medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone and 1% dextrose) and P. methanolica was inoculated at 28°C in a YPAD liquid medium (1% yeast extract, 2% peptone, 2% dextrose and 0.01% adenine).

Cloning and decorating DhIFN-α1: To screen differentially up-regulated genes, subtractive hybridization was performed using a PCR-select cDNA Subtraction Kit (Clontech, Palo Alto, CA, USA). D. hansenii was inoculated in a YM11 medium overnight; after adding NaCl to the medium for a final concentration of 2.5 M of NaCl, the total RNA was extracted after 24 min. The fragment of the DhIFN-α1 gene from a subtraction cDNA library was cloned with the rapid amplification of 5′- and 3′-cDNA ends (5′- and 3′-RACE) using the Gene Racer TM Kit (Invitrogen, USA) by following manufacturer instructions. The sequence of the fragment was queried for similarities through the NCBI database by using BLASTX sequence comparison software (http://www.ncbi.nlm.nih.gov.BLAST). The gene of Ifna 1 (Mus musculus interferon alpha 1, GI: 117168292) encoding the protein of the interferon A1 precursor (NP034632) had the highest similarity by using the queried sequence (the DhIFN-a1 gene). Comparing the cDNA sequence of the Ifna 1 gene and the fragment of DhIFN-α1 gene showed that Ifna 1 has an additional 24 bp at the 5′ end cDNA sequence, which encodes a portion of the signal peptide based on the Target P 1.1. Server prediction (www.cbs.dtu.dk/services.Targetp). DhIFN-α1 was completed using a single peptide sequence and DhIFN-α1 was cloned with PCR using the forward primer of IFN-α1/F, which has an additional EcoRI cutting site and 24 bp; the reverse primer of IFN-α1/R has a BamHI cutting site expressed as follows:

IFN-a1/F: 5’-AGGAATTCAATGGCTAGGCTCTGTG CTTTCCTGATGGT-3’
EcoRI for the *decorated region
IFN- a1/R: 5’-ATGGATCCTTTCTCTTCCCTCAG TCTTCCCAG-3’

BamHI: PCR was performed under the following cycling conditions: 1 cycle of 94°C for 3 min, 30 cycles of 94°C for 1.5 min, 65°C for 1.5 min and 72°C for 1.5 min, followed by 72°C for 10 min and 4°C hold. The presence of the DhIFN-α1 gene was demonstrated through the PCR amplification of a 567 bp fragment by using the described forward and reverse primer. The PCR products were analyzed by using electrophoresis in a 1% agarose gel. The DNA fragment was cloned into a pGEM-T Easy vector (Promega, USA) for sequencing.

Construction of DhIFN-α1 in pMETB expression vector: To examine the DhIFN-a1 functions further, the pMETB vector was transformed using the P. methanolica Expression Kit (Invitrogen, USA). The entire DhIFN-α1 gene was first amplified by performing PCR and using the overexpressed 5’ primer, which introduced an EcoRI site in front of the starting ATG codon and the overexpressed 3’ primer, which introduced a BamHI site before the stop codon. The PCR-amplified product was cloned into the pGEM-T Easy vector to produce DhIFN-α1/pGEM-T. The DNA was transformed into E. coli. After digestion of DhIFN-α1/pGEM-T with EcoRI and BamHI, a 567-bp fragment was gel-purified from agarose by using a WizardR SV Gel and PCR Clean-Up System kit (Promega, USA) and was ligated to a similarly digested pMETB expression vector. The DhIFN-α1/ pMETB fragment was further verified by sequencing.

Overexpression of DhIFN-α1 in P. methanolica: The DNA fragment of the PAUG1/DhIFN-α1/V5/His expression cassette was produced by Pst I digesting the pMETB expression vector and was purified by the Wizard SV Gel and PCR Clean-Up System. The purified DNA fragments of the PAUG1/DhIFN-α1/V5/His expression cassette were added to the P. methanolica electrocompetent cells and then a voltage intensity of 25 μFx0.75 kV (Gene Pulser II Electroporation System, BIO-RAD, USA) was applied through an electro-cuvette. All the cells were then spread on YPAD agar plates containing 1.0 M of NaCl to screen out the transformants of P. methanolica. A portion of absolute methanol was added to the plates to obtain a final concentration of 0.5%. Methanol was added to the plates every day until the colonies grew. First, the transformants were verified using PCR with AUG1 primers by following manufacturer instructions (P. methanolica Expression Kit K1780-01, Invitrogen, USA). PCR analysis was then performed under the following cycling conditions: 1 cycle of 94°C for 3 min; 30 cycles of 94°C for 45 sec; 60°C for 30 sec, 72°C for 1.5 min, followed by 72°C for 10 min and 4°C hold. The PCR amplification of an 875 bp fragment was performed using the forward primer: 5′-CAATTTACATCTTTATTTATTAACG-3′ and reverse primer:5′-GAAGAGAAACATTAGTTGGC-3′. The PCR products were analyzed using electrophoresis in 1% agarose gel.

To determine the growth of the transformant P. methanolica in NaCl and under temperature stress, a single colony of the wild type P. methanolica or the transformant was first inoculated in the YPAD liquid medium overnight at 28°C. An aliquot of 1 mL of each cell culture was transferred to 30 mL of the YPAD liquid medium at 28°C and was shaken until the O.D. 600 had reached 1. The cell culture was then diluted to an O.D. 600 = 0.3 with a fresh YPAD liquid medium supplement. For the drop test under both salt or temperature stress, portions of the 3 μL serial dilution (1x, 10x and 100x) were spotted onto the agar plates, which contained final concentrations of 0.5, 1.0, 1.15, 1.3, 1.5 and 1.8 M of NaCl, respectively for the temperature stress test, the inoculations were 5, 8, 28 , 35, 37 and 40°C, respectively. Because the expression of the insertion gene was induced by the methanol-inducible AUG1 promoter, the plates were with/without MeOH by adding absolute methanol to a final concentration of 0.5%.

Verifying the expression of the DhIFN-α1/V5/His fusion protein: Wild type P. methanolica and the transformant were streaked onto the MD plates and then a single colony of wild type P. methanolica and transformant was transferred into the BMDY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH 6.0), 1.34% YNB, 4x10-5 % biotin and 2% Dextrose) for overnight inoculation at 28°C with shaking. To verify the expression of the fusion protein of DhIFN-α1/V5/His, the cell pellets were harvested by centrifugation and then resuspended in the BMMY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH 6.0), 1.34% YNB, 0.5% methanol and 4x10-5 % biotin); absolute methanol was added to the medium with a final concentration of 0.5% every 24 h for 5 day to induce fusion protein expression. The secreted protein was analyzed using dot blotting. Protein was extracted from individual transformants by using the Total Protein Miniprep Purification Kit (GMbiolab Co., Ltd. USA). Thirty micrograms of protein from the transformants and the wild type were loaded onto the PVDF membrane for immunostaining and the PVDF membrane was incubated with the first antibody of mouse anti-V5 (Nos. 37-7500, Invitrogen, USA) and the second antibody of AP-conjugated rabbit anti-mouse IgG (No. 81-6522, Invitrogen, USA). Finally, BCIP/NBT phosphase (ZYMED, Invitrogen, USA) was added to the PVDF membrane until spots appeared.


RESULTS
Cloning and characteristics of DhIFN-α1: In this study, PCR was successfully employed to clone a fragment of the DhIFN-α1 gene from the subtraction cDNA library of D. hansenii, which was induced by 2.5 M of NaCl for 24 min and inoculated in a YM medium. The DNA fragment of the PCR product was sequenced with a 567 bp open reading frame (Fig. 1a) and it encoded a deduced protein of 189 amino acid residues (Fig. 1b), the sequence of which was a homolog to the gene of Ifna 1 (GI: 117168292) of Mus musculus. Furthermore, the deduced protein sequence was compared with those of related proteins from the EMBL database by using the EMBOSS alignment program; the deduced protein had a 99% similarity to the Mus musculus interferon alpha-1 precursor (NP034632) and therefore, was named DhIFN-α1.

Overexpression of DhIFN-α1 in P. methanolica: After transferring the PAUG1/DhIFN-α1/V5/His expression cassette into P. methanolica, the transformed products were spread onto YPAD agar plates that contained 1 M of NaCl to screen out the successful transformants of P. methanolica that had gained a salt tolerance ability. The DNAs extracted from the transformants of P. methanolica were used as the templates for the PCR reactions with the AUG1 primers. The transformants presented an 875 bp fragment of the PAUG1/DhIFN-α1/V5/His expression cassette (Fig. 2) and DNA from the wild type P. methanolica disappeared from the DNA fragment. The results confirmed that the expression cassette DNA fragment was integrated into the transformed P. methanolica genome.

The function of DhIFN-α1 was further tested by overexpressing the gene using methanol and subjecting P. methanolica to salt stress. The growth of the wild type was not obviously different from the transformant when they were inoculated in the YPAD media content of 0.5, 1, 1.15 and 1.3 M of NaCl with and without methanol. When the NaCl concentration of the YPAD medium was increased to 1.5 M, the growth of the transformants was obviously better than that of the wild type, especially in the presence of methanol in the medium. The wild type and transformants appeared intolerant to 1.8 M of NaCl stress, both with and without methanol (Fig. 3). However, the transformant was able to maintain a better growth rate under high salt conditions, especially in the presence of methanol, which induced the overexpression of DhIFN-α1.

Furthermore, in a growth test under temperature stress, the wild type was not obviously different from the transformant, although it was inoculated in the YPAD liquid media at 8, 28 and 35°C, both with and without methanol. When the temperature of the inoculated YPAD medium was decreased or increased to 5 or 37°C, respectively, the growth of the transformants was obviously better than that of the wild type, especially in the presence of methanol (Fig. 4). The wild type and transformant appeared intolerant to 40°C stress both with and without the addition of methanol. The results indicated that the overexpression of DhIFN-α1 in the transformant of P. methanolica conferred an enhanced temperature tolerance, which enabled the transformant to grow at higher and lower temperatures.

Figure 1(a-b): (a) DhIFN-α1 gene fragment amplified by PCR from the subtraction cDNA library of D. hansenii, (b) The full-length nucleotide sequence of DhIFN-α1 and the predicted amino acid sequence. The start and stop codons ATG and TGA were boxed

Expression of DhIFN-α1/V5/His fusion protein: The white colonies on the MD plate were chosen to verify the Mut+ (methanol usage plus) and MutS (methanol usage slow) transformant colonies of P. methanolica. The transformants were selected to assay the fusion protein expression using dot blotting with an anti-V5 antibody. The transformants were then confirmed as having good expression of the fusion protein that had a V5 epitope in its C-terminal region, resulting in a spot appearing in the dot blotting (Fig. 5). The results confirmed that the methanol-inducible AUG1 promoter drove the expression of the DhIFN-α1/V5/His fusion protein by using methanol.


DISCUSSION
D. hansenii has the unusual ability to grow under high NaCl concentrations13. Prista et al.1 demonstrated that the genetic material from D. hansenii caused a rise in salt tolerance. In this study, the DhIFN-α1 gene was cloned from D. hansenii and P. methanolica was transformed through electroporation. The transformants of P. methanolica were obtained by screening these colonies on plates containing 1 M of NaCl. Successful transformants were confirmed to gain the DNA fragment of the PAUG1/DhIFN-α1/V5/His expression cassette through PCR and the successful expression of the fusion protein of DhIFN-α1/V5/His.

Figure 2: Confirmation of the constructed DhIFN-α1/ pMETB using PCR with AUG1 primer, W: Wild type and T: Transformant

Figure 3: Growth of wild type (W) and transformant (T) of P. methanolica of serial dilutions suspensions were spotted on YPAD medium containing NaCl and MeOH, (a) 0.5 M NaCl, (b) 0.5 M NaCl+MeOH, (c) 1 M NaCl, (d) 1 M NaCl+MeOH, (e) 1.15 M NaCl

Overexpression of DhIFN-α1/V5/His in the transformant was caused by the PAUG1 promoter under the influence of methanol. The fusion protein detected by dot blotting using an anti-V5 antibody was secreted by the transformant of P. methanolica, which demonstrated a higher stress tolerance than the wild type did; hence, we concluded that the DhIFN-α1 protein might enhance the stress tolerance of yeast.

Figure 4: Growth of wild type (W) and transformant (T) of P. methanolica of serial dilutions suspensions were spotted on YPAD medium with and without MeOH under 5, 8, 28, 35, 37 and 40°C temperature stress, (a) 5°C, (b) 5°C+MeOH, (c) 8°C

Figure 5: Dot blot analysis of DhIFN-α1/V5/His fusion protein detected by anti-V5 antibody extracted from transformants (T) of P. methanolica and wild type (W)

The growth test for the wild type and transformants of P. methanolica exposed them to salt or temperature stress (Fig. 3 and 4). The drop tests for growth showed that all P. methanolica transformants were more tolerant to salt and temperature stress than the wild type were, especially in the presence of methanol, which is an inducer that drives the overexpression of DhIFN-α1. However, the DhIFN-α1 gene cloned from the halophilic yeast of D. hansenii demonstrated that overexpression of DhIFN-α1 in the transformants of P. methanolica enhanced its salt or temperature tolerance, enabling the transformant to grow under salt and temperature stress conditions. Most studies have shown that IFNs have great potential as drugs in the treatment of various virus-infected diseases, such as hepatitis C14 and severe acute respiratory syndrome (SARS)15. Most nucleated cells secrete one or more type I IFNs in response to viral infection16. Type I IFNs then induce viral protective responses in neighboring non-infected cells17. Therefore, IFN-α1, a member of the IFN family, is best known for its antiviral activity18. In addition, Beldarrain et al.19 demonstrated that the stabilization of hrIFN-2α is highly dependent on the salt species and its ionic strength. Therefore, it is unremarkable that DhIFN-α1 is one of the major up-regulated genes under salinity stress. However, salt stress in DhIFN-α1 has not yet been studied. Our experimental results showed that the salt stress-induced expression of DhIFN-α1 in D. hansenii provided the transformants of P. methanolica with an enhanced salt tolerance.

To test if the overexpression of DhIFN-α1 enhanced stress tolerance, the overexpression of DhIFN-α1 in the transformants of P. methanolica were inoculated at various temperatures (Fig. 4) in the presence of methanol. The results showed that the transformant of P. methanolica was considerably more tolerant to temperature stress at 5 and 37°C than the wild type was, based on the results of the drop test. Sharma and Kalonia20 reported that the tertiary structure of hrIFN-α2a was significantly degraded with temperatures that increased from 15-50°C. Huang et al.21 showed that the expression of hrIFN-α2a molecules were attractive and increased with temperature; however, the compact conformation of the protein became looser at higher and lower temperatures during bioprocessing. Perhaps most significantly, DhIFN-α and HSP-70 both accumulated in the cells of D. hansenii when they were exposed to salt stress for only 24 min (data not shown); thus, IFN-α and HSP-70 might play a protective role in responding to stress because Heat Shock Proteins (HSP)s are a group of proteins that are rapidly induced when cells are exposed to stress22. Therefore, by rapidly inducing the expression of a wide array of genes in response to cell protein-denaturing stress, IFN-α might be one of the associated genes with high-level expression under salt stress conditions. However, the DhIFN-α1 gene related to salt stress in yeast has yet to be studied.


CONCLUSION
In this experiment, the DhIFN-α1 gene was cloned from the extremely halophilic yeast D. hansenii under salt stress exposure. The transformed yeast of the P. methanolica overexpression provided DhIFN-α1 with a substantial tolerance to salt and temperature. We conclude that the overexpression of the DhIFN-α1 gene in P. methanolica might enhance its stress tolerance.


ACKNOWLEDGMENTS
The authors acknowledge the supports of Tainan District Agricultural Improvement Station, Council of Agriculture, Taiwan Executive Yuan and the Department of Bioagricultural Science, National Chiayi University.


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