For functional binding assays in aqueous systems, only the isolated N-termini of C. oncophora and O. ostertagi depsiphilin (see 7.1) and of the bovine and canine LPH-2 were expressed. Depending on the prediction of conserved domains, the first 443 amino acids (aa) of the depsiphilins and the first 827 aa of the mammalian latrophilins were chosen to be expressed as recombinant proteins in E. coli.
Therefore, the depsiphilin N-termini did not contain the predicted GPS, the transmembrane region and the C-terminus of the receptor. In a previous work on Hc110-R the first 445 aa of this receptor were shown to bind emodepside, α-LTX, and PF1022 A. The recombinantly expressed mammalian latrophilin N-termini included the predicted GPCR proteolytic site (GPS), but also did not contain the transmembrane region and the C-terminus. The transmembrane regions of the receptors cannot be expressed as soluble proteins due to the distinct characteristics of membrane proteins. The protein was examined as described for the isolated N-terminus of Hc110-R in the thesis of SAEGER (2000). Prokaryotic expression was performed using the Gateway® system from INVITROGEN. The system eliminates the need to perform new ligations for each new vector by taking advantage of the site-specific recombination properties of the bacteriophage lambda (LANDY, 1989).
Once, the insert is ligated into a pENTR vector, which contains the site-specific attachment sites attL, the insert can easily be subcloned into the expression vectors.
The expression vectors possess attR sites, which recombine with the attL sites of the pENTRTM vector in a reaction mediated by Gateway® LR ClonaseTM Enzyme Mix. The resulting expression clone has attB sites; the respective by-product with the pENTRTM backbone contains attP sites (HARTLEY et al., 2000).
5.18.1 Attaching Restriction Sites
Prior to cloning the DNA fragments into the pENTRTM 3C vector restriction sites were attached to the ends of the insert to introduce sticky ends for ligation. For this reason a PCR with primers containing specific restriction sites was performed. After PCR the PCR products were analyzed, isolated, cloned into the pCR® 4 TOPO vector, and transformed into One shot® Top 10 chemically competent E. coli as described above (5.7 - 5.10). The aim was to ensure that the DNA fragment was inserted into the
Material and Methods 73
vector in the right orientation. The restriction sites had to fulfill the following requirements: they had to be unique within the pENTRTM vector, and, as far as possible, also within the insert. Furthermore, the reaction setup of the two enzymes for each cloning had to be compatible for a double digest. The templates for PCR were plasmids containing the full-length coding sequences of the respective genes, which were to be expressed. To minimize a start of translation behind the N-terminal tag, leading to untagged recombinant protein, the forward primers changed the start codon of the genes in some cases from ATG to ATC.
5.18.1.1 Primers
The restriction sites attached to the N-terminus of C. oncophora and O. ostertagi were BamHI and EcoRV. The primers for this approach were, for C. oncophora Co Bam ATC F and Oo N-Term EcoRV Re; the reverse primer was originally designed for O. ostertagi depsiphilin. For O. ostertagi the primers were Oo Bam ATC II F and Oo N-Term EcoRV Re. For the N-termini of bovine and canine LPH-2 identical primers could be used, Lat Bam ATC F and Lat Xho Re. These contained restriction sites for BamHI and XhoI, respectively.
5.18.2 Digestion of Plasmids for Cloning into pENTRTM 3C
The resulting plasmids of 5.18.1 were isolated and sequenced as described in 5.13 - 5.17.1. For restriction analysis the respective enzymes for the introduced restriction sites were employed to confirm the expected restriction. For each cloning procedure 400 – 1000 ng of the pENTRTM 3C vector and the insert-containing pCR® 4 TOPO vector were digested. The reaction setup for the digestion of C. oncophora and O. ostertagi depsiphilin (BamHI / EcoRV) and of bovine and canine LPH-2 (BamHI / XhoI) N-terminus is described below.
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5.18.3 Double Digest
Volume Final concentration
ddH2O variable, brings the final volume to 20 µl
plasmid variable 400 – 1000 ng / rxn
Tango 10 X buffer 4 µl 2 X
restriction enzyme I 1 µl 10 U / rxn
restriction enzyme II 1 µl 10 U / rxn
The reaction was incubated for 2 – 4 h at 37° C. Fo r some other applications of double-digests a different 10 X buffer was used, with a final concentration of 1 X. The choice of the buffer system and concentration depended on the manufacturer’s recommendations for the respective combination of enzymes.
5.18.4 Ligation
After digestion, the samples were analyzed on an agarose gel, and the desired bands of the insert and the vector were excised (see 5.7 and 5.8). The gel slices were centrifuged for 10 min at 16 000 x g and the supernatant was saved for ligation.
The ligation setup was as follows:
Volume Final concentration
digested insert 8 µl
digested vector 8 µl
T4 10 X ligase buffer 2 µl 1 X
T4 DNA ligase 2 µl 2 U / rxn
Total volume 20 µl
The ligation sample was incubated at 22° C overnigh t. The next day the T4 DNA ligase was inactivated by heating the mixture to 65° C for 10 min. After chilling, the entire ligation mixture was used for transforming One shot® Top 10 chemically competent E. coli (5.10). The antibiotic to select for plasmids with a pENTRTM backbone was kanamycin. Plasmid DNA was isolated as described in 5.13 – 5.14.
Since the pCR® 4 TOPO vector also carries a kanamycin resistance gene, the plasmids were identified by their restriction pattern. The backbone of the
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pENTRTM 3C vector is approx. 2.4 kb in size, whereas the pCR® 4 TOPO vector is about 3.9 kb. The insert was checked additionally by PCR with gene-specific internal primers.
5.18.5 Gateway® LR ClonaseTM Reaction
The Gateway® LR ClonaseTM Enzyme Mix (INVITROGEN) was used for the LR recombination reaction. The Gateway® LR ClonaseTM Enzyme Mix catalyzes the attL x attR reaction, which is utilized for integrating the insert of a pENTRTM vector into a pDESTTM expression vector. The inserts were subcloned into the pDESTTM 17 vector, which provides an N-terminal His-tag for detection and purification.
The plasmid DNA was diluted with TE buffer (pH 8.0) to 150 ng / µl. The setup was as follows:
Volume
pENTRTM 3C plasmid DNA 2 µl (300 ng) pDESTTM 17 plasmid DNA 2 µl (300 ng) 5 X LR Clonase reaction buffer 4 µl
TE buffer (pH 8.0) 8 µl LR ClonaseTM Enzyme Mix 4 µl
Total volume 20 µl
The sample was mixed well and incubated for 3 h at 25° C, then digested with 2 µl proteinase K solution at 37° C for 10 min. 5 µ l of the LR reaction were transformed into 50 µl Library Efficiency® DH5α® chemically competent E. coli (INVITROGEN) as described in 5.10, except that 450 µl SOC medium instead of 250 µl were added. To ensure that no pENTRTM vector was carried over, the antibiotic was switched to carbenicillin. The pDESTTM 17 vector has an ampicillin and carbenicillin resistance gene, which pENTRTM vectors do not have. The plasmid DNA was isolated as described in 5.13 – 5.14 and confirmed by restriction analysis and gene-specific PCR. The backbone of the pDESTTM 17 vector is 6.4 kb in size. The expression plasmid was transformed into BL21 Star (DE3) One Shot® expression cells (INVITROGEN) using the TSS (Transforming and Storing Solution) transforming procedure (see 5.18.7).
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5.18.6 Empty-vector Control
Some of the expression vectors, e.g. the pDESTTM vectors, contain a ccDB gene, which ensures that no cells containing a vector without insert survive. For propagating and maintaining these vectors, chemically competent E. coli cells of the strain Library Efficiency® DB3.1TM (INVITROGEN) was used. For expression studies an empty-vector control is used as a negative control. For this purpose the ccDB gene was cut out of the vector by restriction enzymes. The digested vector DNA was run on an agarose gel to remove the small DNA fragment containing the ccDB gene. The vector DNA band was excised of the gel and religated in a T4 DNA ligase reaction (5.18.4).
5.18.7 TSS (Transforming and Storing Solution) Transforming Procedure It is not uncommon that an expression clone, which is stored in glycerol stock solution and used for several expression experiments, decreases its protein production rate from experiment to experiment. To avoid this phenomenon, a fresh transformation of expression plasmid was performed for each experiment. The technique applied for this transformation was the TSS transforming procedure (CHUNG et al., 1989). For this transformation method, a special Transforming and Storing Solution (TSS) is used, which can also be used for the storage of E. coli cells.
Initially a culture of untransformed E. coli BL21 Star (DE3) One Shot® cells was grown in LB broth over night and a glycerol stock (5.12) was made. The strain lacks lon and OmpT proteases and RNase E. These genetic characteristics enhance the expression of intact recombinant protein by reducing mRNA and protein degradation.
These cells were used for transformation: for the TSS transforming procedure, 1 ml LB broth per planned expression culture was inoculated with 15 µl of this glycerol stock and incubated at 37° C and 200 rpm u ntil the culture reached an OD 600 = 0.3. For each expression sample 1 ml of culture was transferred into a fresh 1.5 ml tube and centrifuged for 10 min at 380 x g at RT. The supernatant was discarded, and the pellet was resuspended gently in 100 µl ice-cold TSS. The cells were kept on ice and gently mixed with 50 – 100 ng of expression plasmid DNA by gentle stirring. The tubes were kept on ice for 5 – 10 min and then placed at RT (the heat-shock) for another 5 – 10 min. The cells were then put on ice again for 5 – 10 min, then 900 µl of LB broth without antibiotics were added. The sample was
Material and Methods 77
shaken at 200 rpm and 37° C for 1 h. 100 µl of this culture were cultured in 5 ml LB broth containing the appropriate antibiotic overnight at 37° C and 200 rpm.
The antibiotic was carbenicillin for pDESTTM vectors. Approx. 1 – 5 ml of culture were used the following day to inoculate an expression culture of 50 – 250 ml TB broth containing the appropriate antibiotic. The OD600 was adjusted to 0.05 – 0.1 at the beginning of incubation at 37° C and 200 rpm.
5.18.8 Regulation of Expression
Regulation of expression was performed by inducing promotors. The promotors were either controlling the T7 RNA polymerase of the cells or, if included in the vector, directly regulating the transcription level of the gene to be expressed. Therefore, induction of expression depended on cells and vector. As the pDESTTM 17 vector, most of the tested vectors contained a T7 promotor for a T7 RNA polymerase regulated expression of the gene. If these vectors are transformed into cells possessing the λ DE3 lysogen, e.g. BL21 Star (DE3) One Shot® competent cells, expression is inducible: λ DE3 lysogen carries the gene for T7 RNA polymerase under control of the lac UV5 promotor, which can be activated by IPTG. The cascade is therefore: IPTG induces the expression of T7 RNA polymerase, which starts the expression of the desired gene within the expression plasmid. Some other vectors and cells had different induction procedures and will be described in 5.18.10 - 5.18.11.
5.18.9 Inducing Expression Cultures
The expression cultures (5.18.7) were shaken at 200 rpm and 37° C, until they reached an OD600 = 0.4 – 0.8. Then 1 mM IPTG or another appropriate inducer (see 5.18.10 - 5.18.11) was added to start the expression. After 5 – 18 h the cells were pelleted at 1550 x g and 4° C for 20 – 30 min. The pellets could be stor ed at – 80° C for future use.
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5.18.10 Other Bacterial Strains
Other strains of E. coli were tested for their ability to produce the desired proteins in a soluble form. As one of the standard strains of the Gateway® system (INVITROGEN), OneShot® BL21 (DE3) was tested. This strain lacks lon and OmpT proteases, like BL21 Star (DE3) One Shot®, but not RNase E. The expression of T7 RNA polymerase is IPTG inducible.
OneShot® BL21 (DE3) pLysS cells (INVITROGEN) share the advantages of OneShot® BL21 (DE3) cells, but they express T7 lysozyme that reduces the basal expression level and therefore facilitates the expression of potentially toxic proteins.
In BL21-AITM One Shot® cells (INVITROGEN), the expression can be tightly regulated by the araBAD promotor: expression is L-arabinose inducible and can be repressed by glucose. This strain is therefore also applicable for expression of potentially toxic proteins.
Expression was also attempted in Rosetta gami 2 (DE3)TM competent cells (NOVAGEN), which are supplemented with tRNA genes for the codons AGG, AGA, AUA, CUA, CCC, and GGA. These codons are naturally rare in E. coli, but common in some mRNAs coding for eukaryotic proteins. Moreover the strain is able to enhance disulfide bond formation due to mutations in both thioredoxin reductase and glutathione reductase.
5.18.11 Other Expression Vectors
In addition to the pDESTTM 17 vector several other vectors were tested, for their ability to express soluble proteins rather than inclusion bodies. The vector pBAD-DEST 49, belonging to the Gateway® system, provides an N-terminal HP-thioredoxin tag and a C-terminal 6 X His tag. Thioredoxin, as fusion partner, can increase translation efficiency and, sometimes, the solubility of eukaryotic proteins expressed in E. coli (LAVALLIE et al., 1993). pBAD-DEST 49 is regulated by the araBAD promotor and is therefore L-arabinose inducible. To repress basal expression levels glucose can be added, so expression of proteins can be tightly regulated. This might be advantageous for expression of toxic proteins.
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Another vector tested was pRSET ATM (INVITROGEN), which was used previously for the expression of the N-terminus of Hc110-R (SAEGER, 2000). It provides an N-terminal 6 X His tag and gets regulated by the T7 promotor.
As a vector with an N-terminal glutathione-S-transferase (GST) tag, pDESTTM 15 (INVITROGEN) was tested. The GST tag can be used for purification and detection.
Expression is controlled by the T7 promotor as with the pDESTTM 17 vector.
pET-41a(+) (NOVAGEN) was also tested. Inserted genes are expressed as fusion proteins with an N-terminal GST and a C-terminal 6 X His tag. Regulation is by the T7 promotor. The GST tag can be removed.
pCold DNA I (TAKARA) is a vector designed for expression at low temperatures. It provides an N-terminal 6 X His tag for detection and purification. Expression at low temperatures retards the growth and metabolism of bacteria, whereas the expression of the recombinant protein, mediated by the cold shock protein promotor (cspA), is facilitated. The promotor is controlled by a lac operator and IPTG. Expression with pCold DNA vectors can increase the expression of soluble protein, or make expression possible at all.
5.18.12 Coexpression
For potentially improved folding of the proteins other genes were coexpressed with the target gene. Chaperones are involved in the folding process of proteins, therefore their coexpression might help expression of soluble proteins (THOMAS et al., 1997).
The chaperone-coding vectors are described in the appendix (8.4.6). The expression of chaperones and of the desired gene was done on different induction pathways, so the expression levels could be regulated independently. Another attempt was the coexpression of protein disulfide isomerase (PDI) of the canine hookworm A. caninum. The N-terminus of Hc110-R is known to contain a cystein rich region, which might be involved in disulfide bonds. These disulfide bonds likely extend from the N-terminus to the transmembrane region of the receptor, but they might also be important for the tertiary structure of the isolated N-terminus. For this approach expression plasmids of C. oncophora and O. ostertagi depsiphilin N-terminus in a
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pET-41a(+) vector were transformed into One Shot® BL21 (DE3) competent cells, which already contained an expression plasmid of PDI in a pDESTTM 14 vector, using the TSS transforming procedure (5.18.7). The cultures were induced at an OD 600 = 0.4 with 1 mM IPTG and shaken at 37° C and 200 r pm overnight.