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The expression of recombinant proteins in E. coli, a valuable field of biotechnology, is the basic method for protein source in several research models. The Neospora caninum actin fragment had been unsuccessfully expressed in pET28 in E. coli. To overcome this problem, we ligated the expression region of pET28 to pGEM-T Easy Vector. The new hybrid plasmid, named pGEM-pET28, was able to express for the first time an actin fragment from N. caninum that was subsequently purified in a nickel sepharose column. The pGEM-pET28 plasmid kept the N-terminal His-Tag/thrombin/T7/Tag configuration, but it is 1.577 bp smaller than the original pET28 vector and the drug selection is made with ampicillin. The plasmid pGEM-pET28 offers a new option for gene cloning directly in the plasmid for protein expression, demonstrating that the ligation of components from distinct vectors is feasible and advantageous.
Neospora caninum is an obligate intracellular protozoan, strongly correlated to abortion and decrease of fertility in cows, the major intermediate host. The fertility losses caused by the parasite are estimated in more than a billion dollars, disturbing several economies. The members of this phylum are characterized by an active system of invasion. The system is composed of surface and secreted adhesive proteins, which interacts with ligands from host cells and an actin-motor of the parasite. Among the methods applied for studies involving the blockage of the apicomplexan invasion system, heterologous expression in E. coli provides the primary source of potential vaccine antigens, such as AMA-1 (apical membrane antigen) and MSP2 (merozoite superficial protein) from Plasmodium falciparum. This expression system is extensively applied as a source of proteins on laboratory or industrial scale. The most evident features of expression in E. coli are the low cost and high yield of purified proteins, sometimes up to 50% of the cellular protein mass. The induction with IPTG (isopropyl β-D-1-thiogalactopyranoside), based on the lactose operon, allows the expression of proteins after an adequate bacterial growth. Among the several options of expression, the pET plasmids (Novagen), based on pBR322, are one of the most popular in the research and industrial fields. Several approaches based on the pET family (pET 28 and 32) and E. coli (BL21, Rosetta, Rosetta Gami) failed to express fragments of actin from N. caninum by our group. In this work, we ligated the pET28 expression region in the pGEM-T Easy Vector (Promega), generating a hybrid plasmid, named pGEM-pET28. This plasmid succeeded in expressing the actin fragment of N. caninum, representing the first description of actin from N. caninum in its recombinant form. The adaptation of plasmids for expression in E. coli is a valuable tool, which is independent of a commercial organization, offering more options for recombinant expression.
Use of a hybrid plasmid composed of pET28 and pGEM-T Easy for heterologous expression in E. coli.
The expression of recombinant proteins through the application of the pET28 system has been extensively employed. This method is one of the most simple and low-cost procedures for obtaining proteins for diverse applications and is usually the first choice among biotechnologists. This work describes a new plasmid for recombinant expression based on pET28 and IPTG induction, formed by the fusion of the pET28 expression region and the pGEM-T Easy Vector. The hybrid plasmid expressed an actin fragment of N. caninum, an apicomplexan parasite related to abortion in cattle and responsible for significant losses in the cattle industry. The actin has an important role in the gliding and invasion process of apicomplexans in general, and this is the first description from N. caninum, a Toxoplasma gondii-related parasite. Several unsuccessful attempts were previously made by our group to get the recombinant N. caninum actin in expression p lasmids. This problem was solved with the pGEM-pET28 plasmid: the recombinant actin fragment of 22 kDa was promptly expressed in BL21 (Fig. 1C, lane 2), compared to the non-induced control (Fig. 1C, lane 3). The actin fragment was purified (Fig. 1C, lane 4) and the identity was confirmed by mass spectrometry. A fragment (SYELPDGNIITVGNER) covering 16.7% of the expressed protein was identified with a significant score of 92. Rare codons compose approximately 10% of the N. caninum actin (38 rare codons of 375 total codons, according to ATGme software), an important factor for inhibition of protein expression in E. coli. On the other hand, the origin of replication of pGEM (pUC) present in pGEM-pET28 generates higher plasmid copies as compared to pET28, probably compensating the low expression caused by rare codons. Moreover, the reduction of the plasmid size improves transformation efficiency and stability in E. coli, a clear advantage of the pGEM-pET28 compared to pET28. The importance of actin in Apicomplexa has been extensively demonstrated using T. gondii and Plasmodium models. The use of pGEM-pET28 allows future research involving actin of N. caninum, bringing further knowledge of this protein within the phylum and its role in the invasion process. Our work demonstrates that vectors for maintenance of genes such as pGEM are also able to perform the function of expression as pET28, and pGEM-pET28 represents an option for the expression of proteins from deleterious protozoans.
The actin fragment from N. caninum was expressed in E. coli using pGEM-pET28.
Conventional plasmids such as pET28 and pET32 failed to express N. caninum actin. This difficulty was solved with the use of pGEM-pET28. However, the recombinant actin protein was insoluble. Therefore, the next steps should be based on the solubilization of the protein. The addition of solubility-enhancement tags (such as thioredoxin or SUMO proteins) might be considered to achieve soluble and active N. caninum actin.
High copies of pGEM-pET28 in bacteria probably compensate the low expression caused by rare codons present in actin. Thus, pGEM-pET28 is an interesting alternative to express proteins with considerable proportion of rare codons in E. coli.
Ligation of pET28 region in pGEM plasmid
The recombinant expression region of pET28 was amplified by PCR with the primers pET28 forward (TTTCTGCAGCACCACCCTGAATTGACTCTCT) and pET28 reverse (TTTCTGCAGGGATATAGTTCCTCCTTTCAGCA) flanked by PstI restriction sites (in italic). The forward and reverse primers were localized, respectively, at 383 bases upstream of the T7 promoter and 21 bases downstream of the T7 terminator (Fig. 1A). The pET28 fragment was ligated in the pGEM-T Easy Vector (Promega) following the manufacturer’s instructions. The pGEM-pET28 (Fig. 1B) plasmid was cloned in E. coli Top10, extracted with mini-prep (Wizard® Plus SV Minipreps; Promega), and confirmed by sequencing.
N. caninum culture
The tachyzoite forms of N. caninum Nc-1 isolate were cultured in Vero cell cultures as previously described. The purification of tachyzoites was performed by exclusion chromatography in Sephadex G-25 (PD-10 columns; GE). The recovered parasites were counted in a Neubauer chamber after adequate dilution and applied as the source for RNA extraction.
RNA extraction and cDNA synthesis
RNA from N. caninum was extracted following the Trizol protocol (Invitrogen). Freshly purified tachyzoites (1×107) were solubilized in 1 mL Trizol solution and extracted in 1 mL chloroform, followed by centrifugation at 10,000 g for 15 min. The resulting aqueous phase (200 mL) was replaced into a new tube and the RNA was precipitated with 200 mL absolute isopropanol. RNA was pelleted by centrifugation at 10,000 g for 15 min and washed with 500 mL 75% ethanol. The pellet was air-dried, diluted in 50 mL water and quantified in a 260/280 nm spectrophotometer (GeneQuant Pro; Amersham/GE). cDNA was obtained by reverse transcription following the manufacturer’s instructions (GoScript™ Reverse Transcription System kit). Briefly, 500 ng of RNA was incubated with 5 nM of poli-T primer for 1 h at 37°C. The actin fragment of N. caninum (ToxoDB data bank: NcLIV_003440) was amplified from the 604 to 932 bases with the primers forward NcActBamHI (TTTGGATCCACCACCTCCGCC) and reverse NcActHindIII (TTTAAGCTTCCAATACCCTCGT). The restriction sites BamHI and HindIII are shown in italic. The N. caninum actin fragment was cloned in pGEM-T Easy Vector and sequenced.
Expression of actin fragment in pGEM-pET28 plasmid
Both pGEM (with N. caninum actin fragment) and pGEM-pET28 plasmid were treated with BamHI and HindIII. The NcActin fragment, extracted from pGEM, was ligated in the hybrid plasmid. The ligation was electroporated in E. coli Top10, verified with EcoRI treatment, and sequenced. The pGEM-pET28/Actin was electroporated in E. coli BL21 and submitted to expression inducted by 1 mM IPTG. The bacterial pellets were sonicated with 8 M urea and the recombinant actin fragment was purified in an affinity nickel sepharose column (Ni Sepharose 6 Fast Flow; GE). The extracts and aliquots with the recombinant protein were analyzed by SDS-PAGE and stained with coomassie blue G.
The band corresponding to the recombinant actin was excised and subjected to trypsin digestion. Briefly, the sample was washed 3 times in 25 mM NH4HCO3 pH 8.0 in 50% acetonitrile (ACN) for 15 min each wash, dehydrated in 100% ACN, and dried in a vacuum concentrator (Labconco). The gel was resuspended in 25 ng/µL trypsin solution (Trypsin Gold; Promega), incubated for 16–24 h at 37°C, and peptides extracted with 50% ACN and 5% trifluoroacetic acid (TFA). For MS/MS, the peptide solution was diluted 1:1 in α-cyano-4-hydroxycinnamic acid (10 mg/mL in 50% ACN and 0.1% TFA) and applied in a MALDI plate. The calibrant was a polyethylene glycol (PEG) solution diluted in a matrix (1:1). The identification was performed in a matrix-assisted Laser Desorption/Ionization (MALDI TOF/TOF-Bruker). The data were searched against the N. caninum predicted protein database (www.matrixscience.com), using Mascot software version 2.3.02 (Matrix Science). The carbamidomethylation of cysteine and the oxidation of methionine were set as fixed and variable modifications, respectively.
The authors would like to thank CAPES and FAPESP for the PhD fellowship to L.M.P. (2009/07713-7).
The authors would like to thank the Prof. Noberto Peporine Lopes group (Chromatography and Mass Spectrometry Facility from NPPNS-FCFRP/USP) for the technical assistance with the mass spectrometry.