Barondes initially proposed the general name ‘galectins’ in 1994 for all the S-type lectins(1). Galectins are found in multiple intracellular compartments and are secreted into the extracelluar space. There has been an explosion of information on these fascinating proteins in pathological states, particularly inflammation, fibrosis and cancer (2). Galectin3 is capable of binding different types of molecules and exhibits several autocrine and paracrine functions. It can affect cell adhesion, activation, motility, chemoattraction and apoptosis (3-7). Galectin3 expression in cancer has been widely studied; however, there are conflicting results, which make it difficult to come to a general conclusion about the expression profiles of galectin3 in cancer (8). Furthermore, some progress has been made in developing galectin 3 inhibitors as anti-cancer agents(9, 10).
The use of P. pastoris as a cellular host for recombinant protein production has steadily increased. This species can be easily cultured, genetically manipulated, and can reach high cell densities (> 130 g L-1 dry cell weight) on methanol and glucose (11). Other benefits of the P. pastoris system are its strong induciblity and constitutive promoter systems. The methylotrophic yeast Pichiapastoris is widely used as a host system for recombinant protein production (12). Furthermore, it has come into focus for the production of glycol-proteins (with human-like N-glycan structures (13)), as well as several metabolites and recombinant proteins.
The purpose of this study was to express and purify recombinant human galectin 3 in thePichiapastoris expression system.
3. Materials and Methods
3.1. Strains and Plasmids
E.coli strain JM109 was used as a host for cloning. E.coli was grown at 37°C in Luria Broth (LB) media for cloning and Pichiapastoris at 37°C in Yeast-Peptone-Dextrose (YPD) media for expression; neomycin (25 μg.mL-1) was added during growth of strains, containing plasmid. The high-density fermentation media was prepared and vector pcDNA3.1 was used for expression of the target genes. All recombinant DNA manipulation was performed as per the manufacturer instructions (Fermentas).
3.2. Cloning Process
Human galectin 3 transcript variant 1 gene cDNAclone/open reading frame (ORF)clone was procured from Sino biological Inc. on Whatman FTA elute card (Cat. No.: WB120410) and the plasmid was prepared as described by the manufacturer’s instructions. A full-lengthcDNA was amplified with PCR, utilizing P1/P2 (Table 1) as a primer pair in which pGEM-T gene construct served as a template. The 0.8 kb human galectin 3cDNA was subcloned into a yeast vector(pcDNA3.1) and digested with EcoRI andSpeI (Figure 1). The proper orientation of the cDNA insertion was confirmed by restriction enzyme analysis.
4.2. Analysis of Recombinant Proteins
3.3. Primer Sequences
Primers were designed using Primer Express (Applied Biosystems) and, oligonucleotides were synthesized by Bioserve (100 pMol.μL-1). The P1/P2primers were used in standard polymerase chain reactions (PCR) to verify the presence of the galectin 3 gene in the recombinant Pichiapastoris DNA (underlined sections of the primer sequence indicate restriction sites, Hu = human).
3.4. Transformation of P. pastoris by Electroporation
P. pastoris X-33 competent cells were obtained (Invitrogen) and transformed, using the EasySelect Pichia expression kit as per the manufacturer’s instructions; 0.5μg of pcDNA3.1 vector, with a His-tagged galectin 3 insert was added to 100μL of Pichiapastoris X-33 competent cells. Fifteen minutes prior to each transformation, 100 μL aliquot of electrocompetentP. pastoris was kept on ice. The cells were transferred to a pre-cooled electroporation cuvette with a 2 mm interval. Transformation was performed using a BioRadMicropulser, a charging voltage of 2kV and a pulse length of 4ms. Transformation efficiency was recorded and several colonies were chosen for further analysis.
3.5. Galectin 3Purification
Recombinant galectin 3 was purified from Pichiapastoriscultures (5mL) by binding to a HisBind Resin (Qiagen) affinity column. The column was washed with PBS to remove any unbound proteins until the absorbance reached background levels. The bound fraction was subsequently eluted isocratically and the eluted protein fractions containing recombinant galectin 3 were pooled and analyzed by SDS-PAGE. Protein concentrations were determined using Bio-Rad protein assay (Bio-Rad) and bovine serum albumin as the standard, and western blots were used to verify the presence of recombinant galectin 3.
3.6. Analysis of Recombinant Human Galectin 3
This was performed as described previously (14), using human peripheral erythrocytes treated with glutaraldehyde. To prepare the suspension of red blood cells (RBCs) for thehemagglutination assay, heparin-treated human peripheral blood (10 mL) was first centrifuged at 2000×g for 5 minutes. Buffy coat was removed as much as possible and RBCs were washed three times with 50 mL PBS. Next, RBCs were diluted with PBS to obtain 100 mL of an 8% cell suspension. RBC suspension was treated with glutaraldehyde (at a final concentration of 3%) under rotation for one hour at room temperature, followed by washing with 0.0025% NaN3 in PBS. Fixed RBCs were resuspended at 3–4% in PBS–NaN3. Calibration of RBCs was required to obtain the appropriate concentration for lectin-mediated hemagglutination. Serial dilutions of galectins (from 0 to 10 mM) were mixed with the appropriate quantities of RBCs in the wells of a 96-well plate and incubated at 370°C for 30 minutes. When RBCs aggregate (hemagglutination), they spread out like a sheet covering the entire surface of the well. However, RBCs formed very tight button-like precipitations at the bottom of the well, which signifies that there was no aggregation.
Electrophoretic transfer of proteins from the protein gel to the nitrocellulose membrane (BioRad pure nitrocellulose membrane 0.2 μm) was carried out by the semi-dry blotter (Biorad Trans-Blot). For immunodetection, the membrane was incubated in phosphate buffered saline (PBS: 8 g NaCl, 0.2 g KCl, 1.15 g Na2HPO4, 0.2 g KH2PO4 per liter) pH 7.4, containing 5% non-fat milk powder and 1% Tween 20 at room temperature for one hour with gentle shaking. Polyclonal rabbit anti-galectin 3, or mouse anti-galectin 3(Abcam) were diluted (1:5000 or 1:1000) with PBS containing 5% nonfat milk powder and 0.1% Tween 20. The membrane was incubated in primary antibody solution at room temperature for 1.5 hours with gentle shaking. The membrane was then washed in antibody dilution buffer for 3 × 5 minutes at room temperature. Goat anti-rabbit or mouse (for anti-galectin detection) IgG horseradish peroxidase conjugate (Abcam) was used as the secondary antibody (1:1000 dilution) to treat the membrane at room temperature for 1.5 hours followed by 3 × 5 minutes washes in antibody dilution buffer and a rinse in distilled water. Bands were visualized by exposure to UVI-Tech (Gel Imager, UK).
3.8. Identification of p29
The protein band corresponding to 29 kDa was excised from the gels, digested with trypsin (15) and processed for mass spectrometric fingerprinting as described previously (16). In brief, peptide mixtures were partially fractionated on Poros 50 R2 Reverse Phase (RP) microtips and the resulting peptide pools were analyzed by matrix assisted laser desorption ionization-reflectron time of flight mass spectrometry (MALDI-reTOF MS) using a Reflex III instrument (BrukerFranzen). Selected mass values were used to search a protein non-redundant database (NR; National Center for Biotechnology Information) using the Peptide Search (17) algorithm.
4.1. Plasmid and Cloning Process
Plasmid DNA was isolated by the plasmid Mini-Prep (GeneJET Plasmid, fermentas) as per the manufacturer’s instructions. The quantity and quality of isolated DNA was evaluated spectrophotometrically andagarose gel electrophoresis, respectively. The isolated plasmid DNA showed an A260/A280 ratio of 1.8±0.2, indicating relative purity, and 10 μL of the plasmid DNA was used for detection viaethidium bromide stained agarose gels (Figure 2).
The cDNA for human galectin 3 of size 753 bp was obtained from Sinobiologicals (HG 10289-G). PCR amplification of the cDNA was carried out using specific forward and reverse primers (Figure 3). The amplified cDNA was then inserted into the pcDNA3.1 vector using restriction enzymes, EcoRI and SpeI.
4.2.1. HemagglutinationInhibition Assay of Galectin 3
In order to test the bioactivity of galectin 3, hemagglutination assay was performed using human peripheral RBCs. As expected, wells containing RBCs incubated with carbohydrate specific antibodies and 1-10mMgalectin 3 showed sheet like agglutination, where as wells containing RBCs incubated with carbohydrate specific antibodies and 0mM galectin 3 (PBS control), and RBCs incubated with carbohydrate specific antibodies alone (negative control) showed button like sedimentation (Figure 4).
4.3. SDS-PAGE Analysis
Purified recombinant proteins were suspended in a sample buffer (4% SDS, 150 mMTris-HCl (pH 6.8), 20% glycerol, 0.1% bromphenol blue, 1% beta-mercaptoethanol) and subjected to 12% SDS-polyacrylamide gel electrophoresis (PAGE). Various fractions (wash, elution) collected before, during and after protein elution were diluted (1/2) with milliQ ultrapure water before SDS-PAGE analysis. The molecular weight of the galectin protein was shown to be approximately 29 KD, as expected. Gel bands were visualized after staining with Coomassie Blue, as shown by Figure 5.
4.4. Western Blot
In order to assess the immunological relationship between purified galectin 3, the material eluted from the affinity column was submitted to SDS-PAGE and blotted onto nitrocellulose membranes. The 29 kDa protein band strongly reacted with this antibody, indicating that the purified protein is galectin 3. A representative western blot, showing the reactivity of the purified galectin 3 from two different extracts with the A3A12 anti-galectin 3 antibody, is shown in Figure 6.
4.5. Identification of p29
The nine most prominent peaks are labeled in Figure 7; the corresponding m/z values were taken to query the National Center for Biotechnology Information (NCBI) non-redundant protein sequence database (NR; 512000 entries) for pattern matches, using the Peptide Search program. The following criteria were used: six matches out of nine, a mass accuracy of 40 ppm and a maximum of one missed cleavage sites per peptide. Only one protein of less than 100 kDa was retrieved, galectin 3 (matches, 1273, 1324, 1429, 1539, 1626 and 1649; total sequence coverage, 27%). Under these search restrictions, random matches occurred at or below three out of nine. The tryptic digest analysis clearly revealed that the purified protein was indeed galectin 3.
Even though it is of great interest to find reliable, potent and easily available purification methods for galectin 3, the quest has mostly been unsuccessful. Nevertheless, finding quick and efficient methods is of interest, yet the search could benefit from looking past galectin 3. In this study, we constructed a novel shuttle vector in Pichiapastoris and used it for the secretion of human galectin recombinant protein. The galectin was expressed in Pichiapastoris X-33 by growing the yeast culture in YPD medium and purified by nickel-based affinity chromatography due to its His6 tag. In order to elucidate whether galectin 3 expression was differentially regulated throughout the development of yeast cells, western blot analysis was performed. After cell lysis the protein was identified as a single 29 kDa band by 12% SDS-PAGE. Further, Protein bands were excised from the gels, digested with trypsin and processed for mass spectrometric fingerprinting. In brief, peptide mixtures were partially fractionated on Poros 50 R2 RP microtips and the resulting peptide pools were analyzed by matrix assisted laser desorption ionization-reflectron time of flight mass spectrometry (MALDI-reTOF MS). Selected mass values were then taken to search a protein non-redundant database using the Peptide Search algorithm. Mass spectrometric fingerprinting of the purified p29 identified it as galectin3. This indigenously produced recombinant human galectin 3 was evaluated for its biological activity using hemagglutination inhibition assay. In summary, we concluded that, our studies have established an important role for galectin 3 production in Pichiapastoris. In view of the well-established methods available for production, our studies suggest that our method could be easy and reproducible.
The authors would like to thank the management of K L University for their kind encouragement and special thanks are extended to Dr.K.R.S Sambasiva Rao for his helpful suggestions.
All authors participated equally in this study.
There was no conflict of interest.
The study wasself-funded.