Expression of granulocyte colony stimulating factor (GCSF) in Hansenula polymorpha.

BACKGROUND AND OBJECTIVES: During past decades Hansenula polymorpha has attracted global attention for the expression of recombinant proteins due to its high growth rate, minimal nutritional porequirements and use of methanol as a low cost inducer.
MATERIALS AND METHODS: The corresponding nucleotide sequences for the expression of heterologous genes in Hansenula poylmorpha were extracted and assembled in an E. coli vector. The constructed expression cassette included formate dehydrogenase promoter (pFMD), a secretory signal sequence, a multiple cloning site (MCS) and methanol oxidase (MOX) terminator. Zeocin resistance gene fragment and complete cDNA encoding granulocyte colony stimulating factor (GCSF) were cloned downstream of the expression cassette in-frame with signal sequence. Restriction mapping and sequence analysis confirmed the correct cloning procedures. Final vector was transformed into Hansenula and recombinant host was induced for the expression of GCSF protein by adding methanol. SDS-PAGE and immuno-blotting were performed to confirm the identity of r-GCSF.
RESULTS: The expression cassette containing gcsf gene (615bp) and zeocin resistance marker (sh-ble, 1200bp) was prepared and successfully transformed into competent Hansenula polymorpha cells via electroporation. Zeocin resistant colonies were selected and GCSF expression was induced in recombinant Hansenula transformants using 0.5% methanol and an approximately 19kDa protein was observed on SDS-PAGE. Western blot analysis using serum isolated from GCSF-treated rabbit confirmed the identity of the protein.
CONCLUSIONS: Molecular studies confirmed the designed expression cassette containing gcsf gene along with pFMD and signal sequence. The expressed 19kDa protein also confirmed the ability of designed vector in expressing heterologous genes in Hansenula cells.

INTRODUCTION

Expression of heterologous genes is usually performed in prokaryotic host cells especially E. coli which have high growth rates and simple nutritional requirements. On the other hand, the mammalian expression systems attracted much attention for the production of those pharmaceutics with high level of post-translational modifications. However statistical analyses show that in the worldwide biopharmaceutical marketing, beside E. coli and mammalian host cells, low eukaryotic microbial cells including yeasts offer important roles in manufacturing pharmaceutics with low to moderate post-translational modifications such as glycosylation (1). Although the use of the first and the most characterized yeast cell, Saccharomyces cerevisiae, has been FDA approved for the production of recombinant insulin and Hepatitis B vaccines (2) but its drawbacks and limitations such as hyper-glycosylation of the proteins through α1,3 bond of mannose chains to the N terminus of the produced protein has been confirmed to be allergenic.

Hansenula polymorpha (Pichia angusta or Ogataea angusta) is one of the substitutes for the baker’s yeast which can grow in the presence of methanol (MeOH) as the only source of carbon and energy (3). In the presence of glucose, methanol oxidase (MOX) and catalase will be completely repressed whereas shifting to methanol induces their expression (4). Hansenula’s whole genome sequence (5) has provided an excellent opportunity for molecular investigation of this yeast towards efficient expression of biopharmaceutical proteins. The key important features include the presence of inducible promoters in methanol metabolism and thermo-tolerance behavior of Hansenula cells which is critical for large scale fermentation process. Furthermore, it has been shown that the amount of secreted proteins in Hansenula medium is very low which facilitates the purification downstream processing. All mentioned features have made Hansenula as a suitable host for the expression of biopharmaceutical proteins such as interferon alpha-2a and hepatitis B vaccine (6).

The basic elements in Hansenula specific transcriptional unit are: a strong inducible promoter related to the MeOH metabolism, a suitable transcriptional terminator and a selection marker for screening the transformant cells. The latter can be an auxotrophic marker involved in pyrimidine or amino acid biosynthesis such as URA3, LEU2 (7) or a dominant resistance marker. For effective integration of the expression unit into the genome, H. polymorpha-derived autonomously replication sequence (e.g. HARS1), as a replication signal, will be added. Signal sequences can also be fused to the target protein for the secretion in to the medium.

In the present study, the construction of a H. polymorpha expression vector possessing zeocin resistance gene as the screening marker has been described and GCSF as a single chain peptide with minimum glycosylation pattern was expressed to examine the efficiency of the designed expression cassette.

MATERIALS AND METHODS

Construction of Hansenula Expression cassette.

A home made expression cassette in an E. coli cloning vector (pGH, 2900bp) was designed (pYT) containing the following transcriptional elements: pFMD (promoter of formate dehydrogenase) and MOX gene terminator (methanol oxidase, Gen-Bank No. X02425.1). Nucleotide sequence of these elements was extracted from the published genome sequence of Ogataeapara polymorpha (Strain CBS 4732) (5). A 25bp fragment containing restriction sites of unique enzymes including BamHI, SacI, HpaI and BclI was also inserted into the downstream of selected promoter in the synthetic vector in an engineered design to be in one open reading frame with any gene of interest as a cloned gene fragment. In order to construct a secretory expression vector, S. cerevisiae alpha-mating factor (alpha-MF1) leader/signal sequence (267bp) was also inserted at the upstream of the multiple cloning site adjacent to the promoter region. HARS1 (H. polymorpha-derived autonomously replication sequence-1) gene sequence (GeneBank No. X03540.1) was also inserted at 5’ end of the expression cassette which is necessary for insertion of cloned gene into the Hansenula genome. Schematic view of the designed vector is shown in Fig. 1.

Fig. 1.

Schematic view of constructed vector

Cloning of zeocin resistance selection marker.

Due to the prototrophic characteristics of available Hansenula host cell, a selection marker was required to identify recombinant clones. Zeocin resistance (shble, Streptoalloteichus hindustanus bleomycin) gene was amplified through PCR with specific primers (F: 5’-gtagatcttgcggatccccc-3’ and R: 5’- gtagatcttggtctccagcttgc-3’) using Pichia pPICZ A vector (Invitrogen, USA) as a template DNA. Both sense and antisense primers contained 5’ BglII restriction site to facilitate the cloning procedure. PCR cycles were as follows: Initial denaturation at 94°C (5min), 30 cycles of 94 °C (1min), 60 °C (30sec), 72 °C (30sec) which followed with 3min extra extension at 72 °C. The resulting PCR product (approximately1200bp in length) was cloned into BglII site of final expression vector (Fig. 1). The recombinant plasmid was isolated from zeocin resistant E. coli clones grown on agar plates supplemented with zeocin (25μg/ml) and was verified through restriction analysis and PCR.

GCSF encoding sequence.

A 615bp cDNA fragment encoding full length GCSF protein was extracted from Genbank (NM_172219.1) and was optimized for a variety of parameters including codon usage bias, GC percentage, mRNA secondary structures and restriction sites which may interfere with cloning procedures. The optimized DNA fragment harboring BamHI and HpaI restriction sites at 5’ and 3’ ends was chemically synthesized. This fragment was sub-cloned into the BamHI/HpaI linearized constructed Hansenula expression vector (pYT/zeo).

Verification of recombinant pYT/zeo/gcsf vector.

Cloned gcsf and zeocin genes were confirmed with designed primers (for gcsf gene: F-primer: 5’-gtagatcttgcggatccccc-3’; R-primer: 5’-gtagatcttggtctccagcttgc-3’; for zeocin gene: F-primer: 5’-gtagatcttgcggatccccc-3’ and R-primer: 5’-gtagatcttggtctccagcttgc-3’). Restriction digestion analysis using BamHI, EcoRI, HpaI and BglII and final sequencing analysis also confirmed the accurate reading frame in the synthetic expression vector.

Transformation of Hansenula polymorpha.

Prototroph Hansenula polymorpha RB11 strain (efficient in oritidine-5’-phosphate decarboxylase; ura3 positive) was inoculated into non selective YPD broth medium and incubated on a shaker at 37 °C overnight. The culture was diluted with fresh YPD medium to the optical density of 0.8–1.2 at 600nm. Cellular pellet was harvested at 2000 g (5min) and resuspended in 50mM potassium phosphate buffer (pH 7.5) with a final concentration of 25mM DTT. After 15min incubation at 37 °C, cells were washed twice in STM buffer (270mM Sucrose, 10mM Tris-HCl, 1mM Magnesium Chloride, pH 8.5) and resuspended in STM buffer (0.005 initial volume) and kept in ice. The recombinant vector (pYT/zeo/gcsf) was digested at unique EcoRI site located at the most 5’ end of the expression cassette, extracted from agarose gel and transformed into the prepared competent Hansenula cells via electroporation. Transformation was carried out according to the previously published protocol. In brief, 5–10μg linearized pYT/zeo/gcsf expression vector and the backbone vector alone (pYT/zeo) were added to 60μl (approximately 6 X 10 9 cells/ml) prepared competent cells in prechilled 0.2cm cuvettes. Electric pulses (200Ω, 2 kV and 25μF) were given using a BioRad GENE Pulser II and 1ml YPD broth was immediately added to the cell-DNA mixture and incubated at 37 °C for 1 hour. Cells were centrifuged and spread on zeocin free YPD agar plates and incubated at 37 °C for 3–5 days. The grown colonies were selected on YPD plates supplemented with different zeocin concentrations ranging from 400 to 1600μg/ml during several passages and zeocin specific PCR was performed to screen the positive clones for GCSF protein analysis.

GCSF expression induction in Hansenula polymorpha.

Selected recombinant Hansenula clones were screened for the expression of recombinant GCSF. In brief, colonies were inoculated into BMGY (Buffered Glycerol-complex medium) and incubated in a high speed shaker (300rpm) at 30 °C until OD600nm reached to 2–6 (16hrs). After centrifugation (2000 g for 5min), cellular pellet was resuspended in BMMY (Buffered Methanol-complex medium) expression medium and incubated for more growth. To induce protein expression, methanol with the final concentration of 0.5% was added every 24 hours to the medium and incubation at 30 °C extended till 96 hours (4 days). The broth medium was centrifuged at 2000 g (5min) and the supernatant was collected for further analysis.

SDS-PAGE analysis of expressed protein.

To prepare more homogenous supernatant, samples were passed through Centriprep YT50 filters (Millipore, USA) and final samples were run on a 15% SDS-PAGE. The gel was stained with silver nitrate and the expression of GCSF protein was investigated in the samples representing supernatant before filtration (named as crude supernatant or total proteins), filtrate (named as <50kDa) and retentate (named as >50kDa) samples of each control and recombinant Hansenula clones.

Identity confirmation of recombinant protein by western blotting.

Protein samples were run on a 15% SDS-PAGE and transferred to the nitrocellulose membrane. Rabbit anti-GCSF polyclonal serum and HRP conjugated goat anti-Rabbit IgG monoclonal antibody were used as the primary and secondary antibodies in the blotting test and recombinant bands were developed using DAB as the substrate. Commercially available recombinant GCSF produced in E. coli was used as positive control in this test.

RESULTS AND DISCUSSION

Construction of Hansenula expression cassette.

Molecular studies have conducted to develop a wide range expression system applicable for different yeast species including H. polymorpha. The established platform, CoMedTM system, has been applied for comparative expression of several proteins in different yeast host cells (8). Due to the limitations in purchasing native Hansenula strain and its commercial expression vectors, we aimed to construct an appropriate vector for expressing different heterologous genes in the available H. polymorpha cell which was prototroph. Designed expression cassette was constructed in pGH backbone cloning vector, mainly harboring ampicillin resistance gene and ColE1 origin of replication. FMD promoter and MOX terminator fragments (approximately 1000 and 200bp in length, respectively) were integrated in the expression cassette with a multiple cloning site in the middle for cloning of any heterologous gene fragment. Alpha-MF1 signal sequence was also inserted between pFMD and MOX terminator in frame with any cloned target gene in the designed MCS (Fig. 1). A 500bp fragment of HARS1 was also designed at 5’ region of the cassette in order to get more genome integrations and stable transformants. The cassette (HARS1-FMD promoter-MFα1 signal sequence-MCS-MOX terminator) was confirmed by restriction digestion through designed sites at MCS of the constructed vector (Fig. 1).

Previous studies have suggested a variety of selection markers in screening recombinant Hansenula cells. Beside auxotrohic characteristics of the host strains such as URA3, LEU1 and TRP3 markers, use of dominant markers such as kanamycin and phleomycin resistance genes have also been recommended. Zeocin has been categorized as a member of phleomycin/bleomycin antibiotic family and has anti-bacterial as well as anti-fungal activity (9, 10). Previous studies revealed a zeocin sensitive behavior in Hansenula polymorpha in which its 100μg/ml concentration was successfully used in screening Hansenula transformants. An approximately 1200bp sh-ble gene fragment consisting all essential gene fragments encoding zeocin resistance protein, was amplified and the BglII ended fragment was cloned into the same digested expression vector, at the downstream region of the expression cassette. The above cloned fragment contained Transcription Elongation Factor 1 (TEF1) promoter that initiates expression of zeocin resistance gene in yeast cell, EM7 promoter, a prokaryotic promoter expressing zeocin resistance protein in E. coli, sh-ble ORF and 3’ end of Saccharomyces cerevisiae CYC1 gene which is involved in efficient processing and stability of 3’ end of zeocin mRNA (pPICZ A, B and C expression vectors, Invitrogen, USA). This cloning procedure was also confirmed through zeocin specific PCR and BglII digestion (Fig. 2A). The encoded protein binds to the supplemented zeocin in the agar medium and inhibits its DNA cleavage activity and results in zeocin resistance phenomenon.

Fig. 2.

Molecular confirmation of constructed expression vector.

(A) BglII digestion analysis of final vector: #1, 3, 5, 7: undigested expression vectors; #2, 4, 6, 8: BglII digested vectors; M: DNA 1kb-ladder (Thermo Scientific, Cat No. SM0313).

(B) gcsf PCR confirmation of expression vector: #1–6: gcsf positive recombinant expression vectors; #7: PCR negative control; M: DNA 1kb ladder.

(C) SDS-PAGE analysis of GCSF expression: #1–3: Background Hansenula clone (crude supernatant, <50kDa sample and >50kDa sample), respectively; #4–6: Clone #2 (crude supernatant, <50kDa sample and >50kDa sample), respectively; #7–9: Clone#19 (crude supernatant, <50kDa sample and >50kDa sample), respectively; M: Protein size marker (Thermo Scientific, Cat No. 26610).

(D) Immunoblotting confirmation of r-GCSF: #1, 8: Commercial r-GCSF (positive control); #2, 7: Background Hansenula clone (<50kDa sample); #3, 6: Clone#2 (<50kDa sample); #4, 5: Clone#19 (<50kDa sample); M: Protein size marker (Thermo Scientific, Cat No. 26610)(I, II: Rabbit serum samples before and after immunization with r-GCSF protein).

Mature human GCSF is an 18.8kDa protein as a single chain polypeptide consisting 174 amino acids (11). The codon optimized full length GCSF encoding fragment was synthesized and inserted into the final expression vector. Cloning procedure was confirmed through the gcsf specific PCR and restriction digestions representing a 615bp fragment of gcsf gene on agarose gel (Fig. 2B). Sequencing analysis also revealed its correct fusion to the MFα1 signal sequence to secrete the protein into the medium under the control of pFMD promoter.

Recombinant Hansenula transformants.

The constructed plasmid, pYT/zeo/gcsf carrying HARS1 telomeric region and zeocin as a selection marker, was transformed into available Hansenula polymorpha strain. The grown colonies on initial YPD agar plate (in the absence of zeocin) were spread on plates supplemented with initial 100μg/ml concentration of zeocin according to the previous studies (12). Zeocin concentration gradually increased ranging from 100 to 1600μg/ml and final zeocin resistant colonies (on the plate containing 1600μg/ml of zeocin) were stabilized through several passages and genetically confirmed by the presence of approximately 1200bp zeocin resistance gene fragment through specific designed PCR on Hansenula genomic DNA. Our molecular investigation confirmed that the expression cassette and all necessary elements are theoretically located in their correct positions in the designed constructed vector (Fig. 1).

Expression of recombinant GCSF in Hansenula polymorpha.

Methanol utilization as the only carbon source has been described for several yeast strains (13) including Hansenula polymorpha (3) and key enzymes involved in MeOH metabolism have been discovered through extensive molecular studies. In Hansenula polymorpha, MOX and FMD promoters are repeatedly used for the expression of heterologous genes (14–16). These promoters are repressed in the presence of glucose, ethanol or glycerol and adding methanol to the culture medium can induce their activity (17, 18). BMGY and BMMY media used in this study are optimized for the expression of secretory proteins via using phosphate buffer considering a range of pH in which yeast extract and pep-tone may decrease the proteolysis activity against secreted proteins and allow better biomass production. The expression of recombinant GCSF was induced by adding methanol to the liquid medium. Methanol was added to the medium every 24 hours to control the concentration of the inducer in the culture during 96 hours incubation period.

Previous studies have confirmed that the presence of α-MF1 signal sequence originated from S. cerevisiae resulted in the secretion of the recombinant proteins in the culture medium. Culture supernatants were passed through centriprep YM50 filters (with 50kDa cutoff) to collect proteins below 50kDa (mentioned as <50kDa sample in Fig. 2C) including r-GCSF (approximately 19kDa) and collected samples were separately run on 15% SDS-PAGE. A protein of approximately 19kDa was detected on the gel stained with silver nitrate in below 50kDa samples corresponding to Hansenula clones numbered 2 and 19 which was absent in the negative control cell (harboring empty vector) and retentate samples (supernatants above 50kDa named as>50kDa sample) (Fig. 2C).

Identity confirmation of recombinant GCSF protein by immuno-blotting.

Western blotting was performed to confirm the expressed protein as GCSF using a rabbit polyclonal antibody produced against commercially available recombinant GCSF (Filgrastim; Pooyesh Darou Inc., Iran). Membrane was firstly incubated with 1:500 dilution of rabbit polyclonal serum against r-GCSF and then incubated with 1:1000 dilution of the secondary antibody. The result indicated the presence of expected band of 19kDa in the supernatants of confirmed clones in comparison with negative control which was the supernatant of Hansenula cell culture harboring the expression vector (empty vector) (Fig. 2D).

The application of yeast cells in biotechnology refers to many years ago when they were applied in production of breads and alcoholic drinks. Increasing knowledge in their fermentation process extended their application into the production of biopharmaceuticals. These GRAS (generally regarded as safe) organisms and S. cerevisiae in particular, have been well-characterized and several engineered strains have been developed in recent years (19). In the high growing pharmaceutical market E. coli, S. cerevisiae and mammalian cells are the main host cells. Beside high growth rate of E. coli and its simple medium requirements, mammalian cells are the main hosts in producing proteins with huge post-translational modifications (20, 21). On the other hand, yeast cells can serve as the simplest eukaryotic microbes in order to provide high growth rate on simple media as well as specific limited post-translational modifications of specific desired proteins. S. cerevisiae has been served as one of the main yeast cells in biopharmaceutical industry. Its application began with production of recombinant insulin in 1987 and continued by several vaccines and blood factors (20, 22).

Among other yeast strains, methylotrophic yeast Hansenula polymorpha and Pichia pastoris have attracted scientific interests as suitable hosts for the expression of several heterologous genes such as IFN alpha-2a, insulin and hepatitis B vaccines and a few of them have been approved by the FDA (6, 22, 23). This attraction can be due to their advantages in comparison with S. cerevisiae which include the ability to grow in high cell density in simple media and high rate of plasmid integration into the genome (24, 25). On the other hand, the most frequent promoters in S. cerevisiae vectors, GAL1 or GAL10, will be induced by galactose which is an expensive carbon source molecule while methanol acts as a suitable economic carbon source for inducing corresponding strong promoters in other two mentioned yeast cells. In S. cerevisiae, hyper glycosylation through α-1,3-man-nose linkages (50–150 residues per protein) will cause shorter serum half life of produced proteins and immunogenic responses in the body. To overcome this issue, several genetic engineering approaches have been conducted in other yeast strains including H. polymorpha to minimize the level of α-1,3-mannose residues link to the expressed proteins between 8 and 14 (26, 27).

Cloning of gcsf gene was selected to examine the efficiency of constructed vector in expressing heterologous genes. Mature human GCSF protein has two disulfide bonds and a free cysteine in position#17. Without any N-glycosylation site, it has just one O-glycosylation site at Thr-133 position (11) assuming as a GCSF stabilizing moiety (28) via supportive activity for cysteine group (29). This study confirmed the integrity of the expression cassette for the expression of heterologous genes which can be examined for more complicated pharmaceutical proteins.