Research ArticleResearch Article

Expression and Characterization of the Recombinant Human FLT-3 Ligand Extracellular Domain in Pichia Pastoris

Zongtang Huang and Xishan Hao
Chinese Journal of Clinical Oncology December 2006, 3 (6) 400-407;

Abstract

OBJECTIVE The FLT-3 ligand (fms-like tyrosine kinase receptor-3 ligand, FL) is a recently described growth factor affecting early hematopoi-etic progenitor cells. The FL plays a key role in the growth and differentiation of primitive hematopoietic cells. To yield a high-level of recombinant human FL protein, a recombinant Pichia Pastoris (P. pastoris)strain was constructed.

METHODS An artificial expression frame, with the same encoding protein sequence for the FL extracellular domain cDNA, was synthesized by using favored genetic codons of P. pastoris. P. pastoris strain KM71 cells were transformed with the endonuclease Bgl II linearized recombined plasmid, pPIC9K-FL. The plasmid then was linerized in the 5'AOX1 site and integrated into the yeast KM71 genome. KM71 was transformed with pPIC9K plasmids as a control for the production of recombinant protein. Southern blotting and Northern blotting tests were used to screen the genotype of the recombined strain. Biological activity was demonstrated in vitro with culturing of CD34+cells.

RESULTS The recombinant human FL protein expressed into the yeast culture supertant was identified on the basis of its molecular weight and Western blotting analysis. Numerous bands were observed in the 10~100 kDa molecular mass range. SDS -PAGE showed that the expressed product, a 20 kDa protein, was secreted into the medium in the form of a soluble molecule. Western-blot analyses showed good antigenicity and specificity against polyclonal antibodies. A sharp band and a smeared band were observed at a molecular mass of approximately 20 kDa by Western blotting. The recombinant human FL protein was the major protein component observed in the culture supernatant. The highest yield (108 mg/L) was obtained when expression was induced with 0.5% methanol for 96 h. Deglycosylation with PNGase F resulted in a decrease in apparent molecular mass from 20 kDa to 18kDa forming three bands all of which were also detected by rabbit anti-FL antibodies. Culturing of CD34+ cells in the presence of KM71pPIC9K-FL over 7 days increased 2.9 fold, while in the control group they increased only 1.5 fold. The biological assay showed that the expressed product could stimulate the proliferation of CD34+ hematopoietic cells.

CONCLUSION We demonstrated that human FL was secreted into the culture supernatant from P. pastoris, and that this yeast strain was a preferred host for recombinant human FL gene expression. This recombinant strain can provide a convenient process for pharmaceutical application.

KEYWORDS:

keywords

The FLT-3 ligand (fins-like tyrosine kinase receptor-3 ligand, FL) is a recently described growth factor affecting early hematopoietic progenitor cells. The FL plays a key role in the growth and differentiation of primitive hematopoietic ceils. FL promotes the survival, proliferation, and differentiation of hematopoietic progenitors in synergy with other growth factors.[1, 2] Preliminary clinical trials have demonstrated that FL treatment resulted in an antitumor response against human malignances and in development of a variety of bioreactives systems, such as results from studies of the efficacy of ex vivo expanded hematopoietic cells for transplantation therapy.[3-5] Multiple isoforms of FL have been identified.[6, 7] The predominant isoform of FL is the trans-membrane protein that exerts biological activity at the cell surface. Trans-membrane proteins can be cleaved proteolytically to produce a soluble form of the protein that also has biological activity.[8-10]

The success of a commercial process for the production of a recombinant protein requires a nonpathogenic organism amenable to large scale, high cell density fermentation, e.g. Pichia pastoris (P. pastoris). Ideally, while the host organism should secrete relatively few proteins into the culture supernatant that might contaminate or proteolytically degrade the prod-uct, it should have the capacity to secrete large quantities of correctly processed recombinant protein into the culture medium. The methylotrophic yeast, P. pastoris, has been used to develop a system for heterologous gene expression.[11-13] The well-established P. pastoris system has been utilized in industrial-scale production and pharmaceuticals and industrial enzymes produced in P. pastoris have already entered the market.[14-16]

In this work, the FLT-3 ligand extracellular domain was expressed by P. pastoris, and various aspects of the recombinant protein were studied.

Materials and Methods

Materials

All restriction endonucleases, T4 DNA ligase, Taq DNA polymerase and plasmid extraction kits were purchased from Promega. DNA primers were synthesized by Takara, Inc. (Japen). Peptone, yeast extract and yeast nitrogen base were obtained from Difico Laboratory (USA). Polyclonal antibodies against human FLT-3 ligand, the second antibody and recombined protein standard from Escherichia coli (E.coli) were from Jingmei (China). A Western blotting system was purchased from Pierce. The recombinant cDNA of human FLT-3 ligand was synthesized by Takara. All chemicals used in the experiments were of reagent grade.

Strains and plasmids

E.coli DH5α was used for plasmid transformation and preparation. The cloning vector used for synthesis of cDNA was pMD18T. The plasmid vector used for DNA recombinant manipulation and yeast strain KM71 used as the host for synthesis of recombinant FLT-3 ligand were from Invitrogen.

General DNA and protein methods

DNA subcloning, restriction enzyme digestions, gel electrophoresis and DNA fragment recoveries from low melting agarose gels and Southern and Northern blotting were performed according to Molecular Cloning 2nd ed. Polymerase chain reactions (PCR) were performed on the basis of standard protocols. The SDS/PAGE procedure was used for protein separation. Electrophoresis was performed on a 1 mm thick slab, and gels were stained with Coomassie blue. The proteins in the SDS gels were transferred onto a PVDF membrane using an electro blotter according to Molecular Cloning 2nd ed. A Western blotting system of detection was conducted according to its user’s guide. The primary antibody was specifically bound to the FLT-3 ligand in the dilution range 1:2000~1:5000, the secondary antibody showed specificity in the dilution range 1:20000~;1:50000.

Primer synthesis

All primers were synthesized by Takara based on computer analysis. 5’ FL primer: CCG GAA TTC ACT CAA GAT TGT TC; 3’ FL primer: TTG CGG CCG CCT AAG CTG TTG G; 5’ AOX1 primer: CGA CTG GTT CCA ATT GAC AAG C; 3’ AOX1 primer: GCA TGT CAG AAT GCC ATT TG.

Construction of a yeast secretory expression vector for synthesis of the recombinant human FLT-3 ligand

The synthesized cDNA was subcloned in a pMD18T plasmid with an EcoR I site in the 5’ end and Not I in the 3’ end. The recombinant cDNA fragment was then ligated into the same endonuclease site in pPIC9K.

Electrophoretic transformation

The KM71 yeast culture grew overnight to OD600= 1.5. It was centrifuged at 1500 g for 5 min and the pellet resuspend in water. After rewashing the cells in 1M sobitol 3 times, the cells were mixed with linearized recombinant plasmid DNA. The cells were then pulsed according to the parameters for Pichia and the aliquots spread on MD medium.[17, 18]

Growth media and conditions

E.coli DH5α was grown in LB medium. Up to 50 mg/ml ampicillin was added for selective pressure on screening and maintaining the plasmid-containing bacterial colonies. YPD medium was used for yeast KM71 growth and transformation. MD medium was used for selection of His+ transformants. The selected clones were further selected for multiple integrated copies by replating with G418 antibiotic at concentrations of 0.25, 0.5, 1, 2 and 5 mg/mL. For small-scale expression of the selected FL constructs, the clones were first grown in BMGY medium with glycerol as the sole carbon source. For induction of the AOX1 promoter and protein production, the growth medium was changed to the same medium except with 0.5% methanol in place of glycerol as the carbon source.

Endoglycosidase digestion

For N-glycanase digestion, 96-h culture medium was brought to a concentration of 0.5% SDS and 0.1M ß- mercaptoethanol and was denatured by boiling for 3 min. The digestion reaction (typically 60 ¼l of total volume) contained 100 ¼,g total protein and 5 Units of PNGase F (Biolabs) in the reaction solution (0.2 M sodium phosphate, pH 8.6,1.25% NP-40).

Biological activity of the recombinant FLT-3 ligand extracellular domain

CD34+ cells were cultured with supernatant from KM71-FL in synergy with stem cell factor (SCF) and interleukin-3 for 7days. The CD34+ cells were selected using immunomagnetic beads and cultured as the method that had been previously publicated.[19-20]

Results

Construction of the yeast secretion expression vector

The cDNA for the FLT-3 ligand extracellular domain, which includes 156 amino acids, was synthesized following the preferred codons of P. pastoris (Fig.l) based on computer analysis. The P-E-F before the aimed amino acid sequence and under the Glu-Ala repeats would not alter the function of the recombined protein. The secretion plasmids pPIC9K-FL were designed to produce an in-frame fusion between the secretory leader sequence and the FLT-3 ligand extracellular domain coding sequence(Fig.2). The recombinant plasmid was sequenced.

Fig. 1.
Fig. 1.

Synthesized cDNA of the FLT-3 ligand extracellular domain.

Fig. 2.
Fig. 2.

Construction of the expression vector.

Transformation of yeast

The P. pastoris strain KM71 cells were transformed with the endonuclease Bgl II linearized recombined pPIC9K-FL plasmid. Positive transforments that grew on 2 mg/ml G418 antibiotic were subjected to PCR (Fig.3), Southern blotting (Fig.4) and Northern blotting (Fig.5) assays. The KM71 cells were transformed with pPIC9K plasmids as a control for the production of recombinant protein.

Fig. 3.
Fig. 3.

PCR analysis of positive anti-G418 yeast strain. Lane 1: KM71 strain DNA using 5’FL+3’FL primer as the negtive control; Lane 2: Positive strain DNA using 5’FL+3’AOX1 primer; Lane 3: Positive strain DNA using 5’AOX+ 3’FL primer; Lane 4: Positive strain DNA using 5’FL+3’FL;M: 100 bp ladder.

Fig. 4.
Fig. 4.

Southern blot of the selected transforment strain. Lane 1: Not l+EcoR I digested DNA from KM71 strain transformated from pPIC9K plasmid as the control; Lane 2: Not l+EcoR I digested pPIC9K-FL plasmid; Lane 3: Endonuclease Not I digested DNA from the selected transfórmate; Lane 4: Endonuclease EcoR I digested DNA from the selected transfórmate; Lane 5: Not l+EcoR I digested DNA from the selected transfórmate.

Fig. 5.
Fig. 5.

Northern Blot of the selected transfórmate. Lane 1: mRNA isolated from uninduced culture; Lane 2: mRNA isolated from the 24-hour inducement; Lane 3: pPIC9K/FLT-3 plasmid as the control; A: 32P labeled FLT-3 increment as the probe B: 32P labeled 18S rRNA as the probe.

Expression of recombinant human FLT-3 ligand extracellular domain

The recombinant human FL protein expressed into the yeast culture supernatant was identified on the bases of its molecular weight and Western blotting analysis. The results of the SDS-PAGE and the Western blotting are shown in Figs. 6 and 7B. Numerous bands were observed in the 10~100 kDa molecular mass range. A sharp band and a smeared band were observed at molecular masses of approximately 20 kDa by Western blotting. The recombinant human FL protein is the major protein component observed in the culture supernatant. A maximum yield of approximately 108 mg/L was obtained at 96 h after induction.

Fig. 6.
Fig. 6.

SDS-PAGE time-course of expression of FLT-3 ligand extracellular domain in the culture supernatant of recombinant P. pastoris. Samples were taken 24 h (Lane 1), 48 h (Lane 2), 72 h (Lane 3) and 96h (Lane 4) after induction with methanol. Culture supernatant (20 p.l) from each time-point was analyzed by SDS-PAGE. Lane 5 was the molecular markers.

Fig. 7.
Fig. 7.

Western blot showing deglycosylation test of recombinant FLT-3 ligand extracellular domain. A: The protein bands on the SDS gel were visualized by Coomassi blue staining. B: recombinant proteins were partly digested with PNGase F. MW: protein molecular markers; Lane 1: PNGase F partly digested culture supernantant of induced recombinant protein; Lane 2: culture supernantant of induced recombinant protein; Lane 3: E.coli expressed recombinant FLT-3 ligand extracellular domain as the control

Deglycosylation test of recombinant FLT-3 ligand extracellular domain

The results of the SDS-PAGE and the Western blotting are shown in Fig.7. These smears were also detected by rabbit anti-FL antibodies.

Biological activity of the recombinant FLT-3 ligand extracellular domain

Supernatant from the KM71-FL cells was able to induce proliferation of CD34+ cells (Fig.8). After 7 days, the number of CD34+ cells increased 2.9 fold in the presence of KM71pPIC9K-FL, while in the control group it increased only 1.5 fold.

Fig. 8.
Fig. 8.

Recombinant FLT-3 ligand-induced proliferation of CD34* cells. The number of CD34* cells incressed 2.9 fold in the presenceof KM71pPIC9K-FL after 7 days, while in the control group it increased 1.5 fold.

Discussion

A growing focus on gene technology has permeated basic scientific research and industrial biotechnology. Emerging systematic genomics have increased demand for suitable expression systems to analyze and to exploit newly identified target genes. A heterologous gene expression system is chosen with regard to the characteristics and the anticipated applications of the substance to be expressed from the gene of interest. These criteria are predominantly dictated by either an economic rationale, in the case of industrial enzyme production, or by a safety and authenticity aspect, in the case of pharmaceutical production. An ideal system should combine characteristics that suit both sets of demands, thereby providing a low-cost screening and production system for authentically processed and modified protein compounds. In this respect, yeast offers considerable advantages over alternative eukaryotic and prokaryotic systems. Yeast meets safety prerequisites in that they do not harbor pyrogens, pathogens or viral inclusions. Eukaryotic yeasts, including the methylotrophs, are capable of secreting protein and performing secretion-linked protein modification steps, such as processing and glycosylation. As eukaryotic microbial production systems, they combine ease of genetic manipulation with rapid growth to high cell densities on inexpensive media.[21]

Philips Petroleum developed P. pastoris originally as a single-cell protein system in the 1970s, and Pichia has gained widespread attention as an expression system because of its ability to express high levels of heterologous protein. Yeast-based protein expression systems are efficient and economical compared with bacterial mammalian sources. Yeasts grow rapidly and produce proteins using a eukaryotic protein-synthesis pathway, and the costs of media, equipment and infrastructure to culture yeast are lower than that of mam-malian cells. Eukaryotic proteins are produced in an active form and do not need refolding to give activity, as is the case for many eukarotic proteins made in E. coli.[22]

Pichia can be as easily cultured and genetically manipulated as E. coii and it has a eukaryotic secretory pathway similar to that of mammalian cells.[23] Today over 120 heterologous proteins have been expressed in Pichia. Current applications in recombinant pharmaceutical and enzyme industries include, among others, production of insulin-like growth factor, hepatitis B vaccines, human serum albumin and phytase. Proteins can be produced in P. pastoris under fully validated conditions, which is essential for the production of vaccines or therapeutics.

P. pastoris has some major advantages over Saccharomyces cerevisiae (S. cerevisia).[24] The first is the methanol-induced alcohol oxidase 1 gene (AOX1). This is a tightly regulated gene that is repressed in the absence of methanol, and when used to drive heterolo-gous protein production avoids any toxic effects of heterologous protein expression until expression of the product is induced by methanol. In methanol-grown continuous cultures of methylotrophic yeasts, alcohol oxidase can constitute up to 30% of total cell protein. Thus, the synthesis of these key enzymes is subject to a control mechanism determined by the carbon compound in the culture medium. Huge peroxisomes, the cellular compartments for the initial step of methanol metabolism, can take up to 80% of the cell volume under these conditions of high enzyme production. Enzyme synthesis is controlled on the transcriptional level. The strong and regulatable promoter structures of the respective genes have been incorporated as attractive control elements for heterologous gene expression, particularly the promoters of the alcohol oxidase genes AOX1.

The second advantange of using P. pastoris is that it can be grown to higher density than S. cerevisia. Although the amount of protein purified from shake-flask production is often sufficient, for larger amounts of protein, fermentation should be considered. Fermentation should also be considered for a protein that is not expressed well in a shake-flask system, because the culture environment, such as adequate oxygen supply, can be better controlled in a fermentar, making gene induction more effective. Fermentation is essential for secreted proteins, because yields correlate largely with the cell density and fermenter cultures can reach a cell density in the range of 250-400 g/liter of fresh weight. In some cases, switching from shake-flask expression to fermentation can give dramatic increases in yield, with reports of expression level in fermentation being 10-fold higher than in shake flasks. The highest yield reported in P. pastoris is 12 g/liter for tetanus toxin fragment C.[25] Additionally, contamination of a P. pastoris product with endotoxin is not a concern, giving P. pastoris an extra advantage for the production of vaccines or therapeutic drugs compared with bacterial production systems.

Proteins that are secreted from yeast and other eu-karyotic cells through the endoplasmic reticulum usually possess a hydrophobic amino terminal extension, the signal sequence. The first stage in the secreting of foreign proteins from yeast is the use of either the mammalian signal sequence or the use of a yeast signal sequence. Production of protein in P. pastoris is carried out using defined media and recombinant proteins can be secreted into the medium, enabling purification from a material that contains few contaminants. The effort to generate certified high products is low compared with other expression systems, where media components or by-products of the expression organisms’ metabolism can give rise to purification problems. A range of secretion leaders is available to direct translation products into the secretory compartment of the yeasts. In all methylotropha, an element engineered from the S. cerevisiae-derived a-factor gene can be applied for this purpose.[26] Expression vector containing the α-factor secretion signal from S. cerevisia have been used for secretion of FL. We have demonstrated that human FL can be secreted by P. pastoris when the mature FL coding sequence is fused to the secretory leader sequences described. The FL is secreted into the culture supernatant where it represents the major protein component observed in the culture supernatant and where it is easily separated from the cellular components.

Furthermore, in methylotrophic yeasts, vectors used for transformation are stably integrated into the genome, providing for a consistent production process. Transformation procedures for P. pastoris employ methods similarly described for other yeasts, including S. cerevisiae. They can be transformed using wholecell methods or by electroporation. In P. pastoris foreign DNA is targeted to the AOX1 genes. Linearized recombinant DNA is integrated into the host chromosome. Once an expression strain has been generated, it can be stored easily and reproducible production levels can be obtained, because the gene of interest is integrated into the host chromosome rather than being contained in a plasmid.

The post-translational modification of P. pastoris is a eukaryotic system and enables the production of several functional mammalian glycoproteins, which are essentially indistinguishable from the original. P. pastoris is especially suitable for expression of proteins that can only expressed as insoluble inclusion bodies in bacteria, or that require post-translation modification. [27] The recombinant FL, which was secreted by P. pastoris, was a glycoprotein identified by deglycosylation.

The recombinant FL promotes proliferation of hematopoietic progenitor CD34+ cells in synergy with other growth factors. We demonstrated the secretion of human FL into the culture supernatant from the yeast P. pastoris, and this yeast strain was a preferred host for recombinant human FL gene expression. Employing this recombinant strain will provide a convenient means for pharmaceutical application.

  • Received September 15, 2006.
  • Accepted December 12, 2006.

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