The phytopathogenic fungus Venturia inaequalis causes scab of apple. Once this fungus penetrates the plant surface, it forms a specialized body called a stroma between the inner cuticle surface and the epidermal cell wall. A novel Venturia inaequalis 5704 (Cin3) and three expressed sequence tags (ESTs); 38, 6987, and 4010 are strongly up-regulated in the early stages of infection. The CIN3 and three ESTs using two vectors pMAL-c2 and pET 21 were expressed in Escherichia coli. Recombinant proteins expression, solubility and yields were analyzed. 38, 5704 (Cin3) and 6987 re- combinant proteins were expressed in soluble form and while 4010 was expressed in inclusion bodies. Re- solution on native-PAGE, the recombinant proteins; 38, 5704 (Cin3), 6987 were shown to be present in dimmer, tetramer and polymer. A method was de- veloped, consisting of induction of expression at va- rious temperatures, and using enriched broth with 4% glycerol together with slow shaking, led to a decrease in concentration of nascent polypeptide and production of soluble recombinant proteins of; 38, 5704 (Cin3), 6987 and 4010. Resolution on native- PAGE, the recombinant proteins were shown to be present as monomer.
Ventura inaequalis (Cooke) Wint. causes black spot or scab of apple (Maluspumila (Mill.) Henry) [
Differentiation of sub-cuticular hyphae and stromata can be simulated by growing V. inaequalis in vitro on cellophane discs [
A reliable and efficient protein expression system was needed to make V. inaequalis proteins in their “native” form for protein crystallization and for production of monoclonal antibodies that would provide powerful tools for characterization of V. inaequalis genes function and protein interactions in signal transduction. In this report, we describe a strategy for expression and purification of the four recombinant proteins of V. Inaequalis: 38, 5704 (Cin3), 6987 and 4010.
pMAL protein expression and purification system was purchased from New England BioLabs. Bacterial strain BL21 (DE3) was purchased from Novagen (Madison, WI, USA.). DNA manipulations were carried out by using standard procedures [
The pETM plasmids from EMBL have been used as the back bone to construct the suitable expression vectors for our E. coli expression system. All of the vectors are IPTG inducible and Kanamycin resistant with a general formula of X-Y-rTEV-MCS-Z (see
http://www.emblhamburg.de/~geerlof/webPP/genetoprotein/clo_ vector/our_Ec_vectors.html). The modifications of these vectors were carried out using the method reported previously previously [
followed by R-tag at the 30 of the new MCS was incurporated using PCR with primers 5’CATGCTCGAGGAAAACCTGTACTTTCAGGGTCCGGATCAGTATGAATACAAATATCCGTAGTGAGATCCGGCTAACAAAGC-3’ and 5’-GCGATCCCCGGGAAAACAGCAT-3’. An in vitro biotinylation site, GlyGlyGlyCys peptide followed by two stop codons, was further incorporated into TrxA fusion vector at 3’ of the MCS using primers 5’CCGCTCGAGGGCGTGGCTGCTAGTGAGTCCGGCTGCTAAC-3’ and 5’-GCGATCCCCGGGAAAACAGCAT-3’.
The sequence of 38, 6987, 5704 and 4010 genes are: MSRPGTDPNQDPAYVPAAGDGSYTVCTPYDMPGICKRYKKDGTATKEVAKCRSASQCWVNG NGCVMVGNGFANCSG,MILEPKAGLGEIRSLEQRPAFSDLKASLSPKSACDKEDESDCTAFCKANDQTATCTAAGNKITCSCKGGKSESNCEERCLLCMPGKSQLEEASLLFKQGGSKLFKEDL, DKQEGSDAVNTRYLAKRQSDIPTYHLWDEEKESGVKAAYKVVDGQEVKGQVEKRQSDAPYYHKLWDEENGAVAKAAYRAVDGQKVKGQVEKRQSDAPYYHKLWDEENGAAAKAAYRAVD, and SQAQAPLQQPITQSTDLSPRILPVVTSNELLSLHR KLIEIESISGNEKPVGEWLKGYLEAKNLTVELQEVEEGRYNVFAYPGTERKTKVLVSSHIDTVPPYWSYERKTTDGVDEIWGRGSVDAKACVASQIIAVLDLLESGKHNLPSDALSLLFVIGEEVGGEGMRFFSDRKPTNYSAIVFGEPTEGKLVAGHKGMIGVKLNITGKAAHSGYPWLGISANNVLVQALSIVLALEKDDLPGSKKFGKTTVNIGRVSGGLAANVVAESSKADIAIRIAGGSPEEINKIITKALQPLKEETEKVGGIFELQWSKRAYGTVDIDTDVEGFDTITVNYGTDVPNIEGDHKRYLYGPGSIFVAHSDHEHLAVSELEQSVLDFQKIILAQF respectively.
E. coli BL21 (DE3) clones were cultivated at 37˚C in Luria-Bertani (LB) medium containing Kanamycin (50 μg/mL). When OD600 reached 0.8 - 1.2, isopropyl-β-Dthiogalactopyranoside (IPTG) was added to the medium at a concentration of 0.3 mM, and the cells were continuously cultivated from 3 to 24 h at 10˚C, 15˚C, 25˚C, or 37˚C. Samples (10 mL) were collected at specific time intervals after induction. Bacterial growth was determined by monitoring the OD600 spectrophotometrically The cells harvested from 10 mL of each of the cultures, were washed with 2 mL of 20 mM phosphate buffer (pH 7.5), and then were resuspended with 1ml of 20 mM phosphate buffer (pH 7.5). The suspensions were sonicated on ice-bath and then centrifuged at 20,000 × g for 15 min at 4˚C. The supernatants were taken as soluble fractions. Proteins in the precipitates were suspended in 1 ml of 20 mM phosphate buffer to obtain insoluble fractions. The soluble and insoluble fractions were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were visualized by staining with Coomassie blue-R. Protein concentration was estimated using image densitometry software ImageJ (http://rsb.info.nih.gov/ij/). The appropriate condition for further production of soluble fusion protein was selected based on the highest concentration of fusion protein obtained.
For production of soluble recombinant proteins, 2 - l flask containing 1000 mL of rich YT medium (30 g Tryptone and 15 g Yeast extract) supplemented with Kanamycin (50 μg/mL) and glycerol (4% v/v) were inoculated with 10 mL of overnight culture of E. coli BL21 (DE3) containing the MBP or Trx A tag fusion proteins. The cells were grown at 37˚C, with shaking (250 rpm) to an absorbance (600 nm) of 0.7 - 1.2, and then the medium was cooled to appropriate temperature in a water bath. IPTG was added to the medium at a concentration of 0.3 mM and the cells were continuously cultivated from 3 to 24 h at proper temperature with slow shaking (60 RPM) or static condition. Cells were collected by centrifugation at 6000 rpm for 10 min (Sorval RC5B). The pellets were resuspended in 40 mL of TrisHCl buffer pH 7.8 (20 mM Tris-HCl, 1 mM EDTA, 1 mM PMSF). The cells were disrupted by sonication on ice bath (20 × 10 s). Cell lysates were centrifuged at 16,000 rpm for 20 min, the supernatants collected and placed on ice. For purification of the MBP-tag fusion proteins, the soluble fraction containing MBP-tag fusion protein was loaded on an amylose affinity column, washed with six column volumes of 20 mM Tris, pH 7.8, and 100 mM NaCl, then with three column volumes of 20 mM Tris, pH 7.8, and 10 mM NaCl. Elution was performed with the same buffer supplemented with 10 mM maltose. Eluted recombinant proteins were further purified by employing DEAE column chromatography. The eluent was loaded into a 5 mL HiTrap DEAE column (GE Healthcare Life Sciences) equilibrated with 20 mL of sample buffer, the column washed with 20 mL sample buffer, and the protein was eluted at 2 mL/min, on an AKTA Prime (Amersham Biosciences) with a gradient of 0 - 1 M Nacl. A flow through that contain more than 50% - 80% of un-bound recombinant proteins was concentrated into 200 µL by using Vivaspin 20 ml Centrifugal Concentrators (Cole-Parmer 625 East Bunker Court, Vernon Hills, Illinois, USA). The recombinant protein was denatured by addition of 2 ml of buffer A (8 M urea, 100 mM Tris-HCl, 5 mM EDTA, 10 mM dithiothreitol, 1.5 mM reduced glutathione, 0.2 mM oxidized glutathione, pH 8.4). The insoluble material was removed by centrifugation using micro-centrifuge (14,000 rpm for 10 min). The supernatant was collected and refolding of proteins was carried as described previously [
All of the soluble proteins were confirmed by the native PAGE. Soluble aggregates stay on the top of the gel and do not enter the body of the gel whereas non-aggregated proteins enter the body of the gel.
Protein samples were analyzed for purity and checked for degradation using SDS and native polyacrylamide gels. Samples were resolved on a 7.5% denaturing and native gel. Protein bands were either stained with Coomassie blue R or electro-transferred from an unstained gel onto a nitrocellulose membrane (Trans-Blot Transfer, Bio-Rad Laboratories, CA, USA) for Western blot analysis. The membrane was blocked in 0.5% I-block (Tropix, Bedfod, MA, USA) in phosphate-buffered saline containing 0.1% Tween 20 (PBST) for 2 h at room temperature and incubated for 1 h in 1:4000 dilution of peroxidase-labeled Anti-His6 (Roche # 1 965 085). The membrane was washed three times in PBS and developed using the ECL system (NEN Western Lightning Plus).
His-tag, MBP-tag or Trx A tag recombinant proteins were separated on SDS-polyacrylamide gels and stained with Coomassie blue R. Protein concentration was estimated using image densitometry software ImageJ (http://rsb.info.nih.gov/ij/). Briefly samples were run on an SDS-polyacrylamide gel, Coomassie-stained, imaged on an HP Scan Jet 4300C and analysed with ImageJ software. The blots were processed with the ImageJ program to quantify relative signal intensities of his-tagg positive areas of the membrane.
Protein concentration was also determined using the BCA assay (Pierce chemicals) using bovine serum albumin as standard.
The objective of this work was to develop a protocol for production of pure soluble full-length of four V. inaequalis ESTs to be used for immunization and production of monoclonal antibodies, by insertion of the genes; 38, 6987, 5704 (Cin3), and 4010 into two vectors [(pETM-41 (MBP fusion), and pETM-20 (Trx A fusion)] followed by expression in E. coli. To obtain strong immunological responses with small antigens such as EST proteins, it was necessary that these antigens be expressed as soluble proteins with fusion tags that were strongly immunogenic. We adopted MBP as the fusion partner for this purpose. Other fusion tags that could be cleaved by specific proteases, together with a variety of peptide tags suitable for either purification or attachment to a solid surface for protein arraying were also examined. Fusion tags have been reported to protect their fusion partners from intracellular proteolysis [7,8] and to have the ability to enhance the solubility of their fusion partners [
The C-terminal GlyGlyGlyCys tag constructed together with N-terminal Trx A has proven to be excellent for biotinylation of V. inaequalis ESTs fusion proteins for use in cell fusion and screening experiments. Furthermore for detection and purification, use of a small peptide R-tag has proven extremely useful. R-tag does not occur in plant, E. coli or mammalian expression hosts and its use has minimized cross-reactions. We have previously reported the use of R-tag and an immuno-affinity matrix coupled with its monoclonal antibody for purification [
All of these constructs incorporate different combinations of tags for different affinity purification procedures, and allow for flexibility during purification as well as applications for immunization, hybridoma production and screening for monoclonal antibody.
Proteolytic cleavage sites were introduced between the fusion tags and target protein in order to remove the fusion tags when this was needed. It has been reported that some commercial proteases used for removal of fusion tags, such as enterokinase and Factor Xa, cleave fusion proteins at non-canonical sites and often result in the degradation of the target proteins [10,11]. The highly site-specific rTEV protease [
For production of soluble proteins, six flask of 200 mL (numbered from 1 - 4) each contain 50 mL YT medium containing Kanamycin (50 μg/mL) (see Materials and Methods) were inoculated with E. coli BL21 (DE3) clone and cultivated at 37˚C. When OD600 reached 0.8 - 1.2, the culture media (1 - 4) were cooled to 10˚C, 15˚C, 25˚C, and 30˚C in water bath, respectively, then isopropyl-β-D-thiogalactopyranoside (IPTG) were added to the media at a concentration of 0.3 mM. The cells were continuously cultivated from 3 to 24 h at 14˚C, 25˚C, 30˚C, or 37˚C. Expression at various temperatures, using an optimal concentration of IPTG was tested for each recombinant protein. The test of solubility was carried out
by centrifugation of the lysates followed by resolution by SDS-PAGE. The recombinant proteins; 4010 (TrexA-tagged or MBP-tagged) at 37˚C were expressed in the inclusion body while the recombinant proteins 38, 6987 and 5704 (Cin3) present in the supernatants as resolved on SDS gels to give a protein of the correct size (
Expression at 30˚C showed that 38, 6987 and 5704 (Cin3) present in the supernatant as resolved on SDS gels to give a proteins of the correct size. The recombinant protein 38 enter the body of a 7.5% native PAGE and shown to be present as monomers, while the recombinant protein 6987 and 5704 (Cin3) protein enter the body of a 7.5% native PAGE present as monomer, dimer and tetramer (results not shown but similar to
Induction at 24˚C resulted in production of soluble 6987 and 5704 (Cin3) recombinant proteins and shown to be present as monomers (results not shown) and induction at 14˚C resulted in production of approximately 30% of soluble recombinant protein of 4010. Separation of soluble protein fraction of 4010 from soluble microaggregate, was carried out by employing DEAE column chromatography. The eluent from amylose affinity column was loaded into DEAE column. Approximately 30% of the recombinant protein was bind into the column and the rest of the protein appeared in the flow through. The unbound protein was collected and concentrated into 200 µL by using Vivaspin centrifugal concentrator. The recombinant protein was denatured and refolded as de scribed previously [
the His-tag or rab-tag showed one major bands of the fusion protein (
Production of insoluble fusion protein can be attributed to overproduction of the fusion protein itself. It has been observed that the increase in concentration of nascent polypeptide chain is sufficient to induce the formation of inactive aggregates even upon overexpression of homologous cytosolic proteins [
Also folding failures of about 60% recombinant proteins produced in E. coli is generally attributed to a limitation in the cell concentration of folding supporter elements, which cannot process the newly synthesized aggregation prone polypeptides. This assumption is physiologically supported by the overexpression of chaperone genes, in particular of chaperone genes from the heatshock protein family, in response to recombinant protein overproduction [23-25]. We therefore developed a method that involved induction of expression of recombinant proteins under changing temperature conditions and using rich broth that contain glycerol (4% v/v) with low aeration (see material and methods). Also, before induction, the cultures were cooled to required temperature followed by induction and incubation of cultures at low temperature with slow shaking (10 - 40 RPM) to decrease aeration. Andersen and Meyenburg [
Du Xiu-bol et al. [
aDetermined by using a BCA assay; bDetermined by image densitometry software (ImageJ: http://rsb.info.nih.gov/ij/) of SDS-polyacrylamide gel (data not shown).
We thank Christopher A. Kirk from Plant and Food Research Institute, Palmerston North, NZL for expert technical assistance.