Expression vectors (see additional file 2 : Table of vectors used in this study) were made by standard molecular biology techniques.
Expression vectors for Erv1p, mature DsbC, mature PhoA, mature AppA, mature PDI and mature BPTI were constructed previously [12 (link),30 (link),31 (link)]. The gene for mature MBP (Lys27-Thr392) was amplified by PCR using a colony of E. coli XL1-Blue as a template. The genes for the kringle 2 and protease domains of human tissue plasminogen activator (vtPA; Gly211-Pro562), mature human resistin (Lys19-Pro108), mature human CSF3 (Ala30-Pro207), mature human BMP4 (Pro294-Arg408), mature human Ero1α (Glu24-His468), human enterokinase light chain (Ile785-His1019), mature human interferon α2 (Cys24-Glu188) and mature human interleukin 17 (Gly24-Ala155) were amplified using IMAGE clones as templates.
Mature BPTI, human vtPA, human tissue factor luminal domain, human CSF3, human Ero1α, human enterokinase light chain and human interferon α2 were cloned into a modified version of pET23a which includes an N-terminal his-tag in frame with the cloned gene and an additional SpeI site in the multi-cloning site between the EcoRI and SacI sites. The resulting gene products include the sequence MHHHHHHM- prior to the first amino acid of the protein sequence.
Polycistronic vectors were constructed by taking fragments encoding the folding factors from the pET23 based constructs which include the ribosome binding site e.g. XbaI/X fragments and ligating them into the SpeI/X cut plasmid encoding the protein of interest (where X is an appropriate restriction site found in the multi-cloning site after SpeI and not found in either gene e.g. XhoI). After a single such ligation this generates a plasmid that contains a single transcription initiator/terminator and hence makes a single mRNA, but has two ribosome binding sites and makes two proteins by co-expression from two translation initiation sites (Figure2A ). This ligation results in the loss of the original SpeI site. Transfer of a SpeI site after the second gene into the new vector allows a third gene to be cloned by the same method resulting in a tricistronic vector which makes three proteins from a single mRNA. Note that to clone mature human PDI into these polycistronic vectors silent mutations were made in the two internal XhoI sites in the gene.
A variety of MBP-fusion protein constructs were made. All were based on the expression of mature MBP (Lys27-Thr392) cloned NcoI/BamHI into pET23 d. At the 3' end of the MBP gene a variety of flexible linkers were introduced. These were: i) NSSSNNNNHM; ii) GSGSGSGSGSIEGRGSGSGSGSGSHM, allowing cleavage by Factor Xa and iii) GSGSGSGSGSDDDDKHM, allowing cleavage by enterokinase, with all three having a NdeI restriction site encoded by the terminal His-Met to allow construction of the fusion protein. Several of the proteins of interest were tested in two or more of these MBP variants and for most no significant differences were observed between the variants. vtPA was tested in all three variants and the activity obtained with the first variant was significantly lower (circa 30%) than for the other two under all conditions tested. Due to the relative costs of the proteases the Factor Xa containing linker version of the MBP-fusion protein construct was our version of choice here (denoted MBPx) and all fusion protein data shown here is that vector unless otherwise stated.
The generation of the pre-expression vectors was complicated due to the lack of suitable restriction sites between our chosen host vector (pLysS), the pET23 based vectors we had already cloned our folding factors into and the pBAD 102/D-TOPO vector (Invitrogen) from which we cloned the arabinose promoter. pLysS was chosen as the backbone for the pre-expression vector as it, or a derivative of it is already in all of our host strains and it is fully compatible with co-transfection with many commercial expression vectors, not only pET23. We would also have liked to clone this system into pLysSRARE, but lack of available information on this vector hampered us. pLysS was modified with an NsiI site added at position 3071 and an AvrII site added at position 3578. pBAD 102/D-TOPO was modified by adding an AvrII site at 1089 a XbaI site at 316 and a XhoI site at 796. The genes encoding for Erv1p or Erv1p and mature DsbC or Erv1p and mature PDI were digested XbaI/XhoI from a pET23 based plasmid and cloned into the same sites in the modified pBAD102/D-TOPO. Note that this takes the ribosome binding sites from pET23 and removes a fragment from pBAD102/D-TOPO that includes the ribosome binding site, his-patch thioredoxin, enterokinase site, TOPO sites and V5 epitope. The genes of interest were then cloned NsiI/AvrII from this vector into the modified pLysS. This results in a modified pLysS that includes not only the genes of interest under the pBAD arabinose promoter, but also the araC gene for regulation.
Mutagenesis of plasmids was carried out using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturers' instructions.
All plasmid purification was performed using the QIAprep spin miniprep kit (Qiagen) and all purification from agarose gels was performed using the Gel extraction kit (Qiagen), both according to the manufacturers' instructions.
All plasmids generated were sequenced to ensure there were no errors in the cloned genes (see additional file2 : Table of vectors used in this study for plasmid names and details).
Expression vectors for Erv1p, mature DsbC, mature PhoA, mature AppA, mature PDI and mature BPTI were constructed previously [12 (link),30 (link),31 (link)]. The gene for mature MBP (Lys27-Thr392) was amplified by PCR using a colony of E. coli XL1-Blue as a template. The genes for the kringle 2 and protease domains of human tissue plasminogen activator (vtPA; Gly211-Pro562), mature human resistin (Lys19-Pro108), mature human CSF3 (Ala30-Pro207), mature human BMP4 (Pro294-Arg408), mature human Ero1α (Glu24-His468), human enterokinase light chain (Ile785-His1019), mature human interferon α2 (Cys24-Glu188) and mature human interleukin 17 (Gly24-Ala155) were amplified using IMAGE clones as templates.
Mature BPTI, human vtPA, human tissue factor luminal domain, human CSF3, human Ero1α, human enterokinase light chain and human interferon α2 were cloned into a modified version of pET23a which includes an N-terminal his-tag in frame with the cloned gene and an additional SpeI site in the multi-cloning site between the EcoRI and SacI sites. The resulting gene products include the sequence MHHHHHHM- prior to the first amino acid of the protein sequence.
Polycistronic vectors were constructed by taking fragments encoding the folding factors from the pET23 based constructs which include the ribosome binding site e.g. XbaI/X fragments and ligating them into the SpeI/X cut plasmid encoding the protein of interest (where X is an appropriate restriction site found in the multi-cloning site after SpeI and not found in either gene e.g. XhoI). After a single such ligation this generates a plasmid that contains a single transcription initiator/terminator and hence makes a single mRNA, but has two ribosome binding sites and makes two proteins by co-expression from two translation initiation sites (Figure
A variety of MBP-fusion protein constructs were made. All were based on the expression of mature MBP (Lys27-Thr392) cloned NcoI/BamHI into pET23 d. At the 3' end of the MBP gene a variety of flexible linkers were introduced. These were: i) NSSSNNNNHM; ii) GSGSGSGSGSIEGRGSGSGSGSGSHM, allowing cleavage by Factor Xa and iii) GSGSGSGSGSDDDDKHM, allowing cleavage by enterokinase, with all three having a NdeI restriction site encoded by the terminal His-Met to allow construction of the fusion protein. Several of the proteins of interest were tested in two or more of these MBP variants and for most no significant differences were observed between the variants. vtPA was tested in all three variants and the activity obtained with the first variant was significantly lower (circa 30%) than for the other two under all conditions tested. Due to the relative costs of the proteases the Factor Xa containing linker version of the MBP-fusion protein construct was our version of choice here (denoted MBPx) and all fusion protein data shown here is that vector unless otherwise stated.
The generation of the pre-expression vectors was complicated due to the lack of suitable restriction sites between our chosen host vector (pLysS), the pET23 based vectors we had already cloned our folding factors into and the pBAD 102/D-TOPO vector (Invitrogen) from which we cloned the arabinose promoter. pLysS was chosen as the backbone for the pre-expression vector as it, or a derivative of it is already in all of our host strains and it is fully compatible with co-transfection with many commercial expression vectors, not only pET23. We would also have liked to clone this system into pLysSRARE, but lack of available information on this vector hampered us. pLysS was modified with an NsiI site added at position 3071 and an AvrII site added at position 3578. pBAD 102/D-TOPO was modified by adding an AvrII site at 1089 a XbaI site at 316 and a XhoI site at 796. The genes encoding for Erv1p or Erv1p and mature DsbC or Erv1p and mature PDI were digested XbaI/XhoI from a pET23 based plasmid and cloned into the same sites in the modified pBAD102/D-TOPO. Note that this takes the ribosome binding sites from pET23 and removes a fragment from pBAD102/D-TOPO that includes the ribosome binding site, his-patch thioredoxin, enterokinase site, TOPO sites and V5 epitope. The genes of interest were then cloned NsiI/AvrII from this vector into the modified pLysS. This results in a modified pLysS that includes not only the genes of interest under the pBAD arabinose promoter, but also the araC gene for regulation.
Mutagenesis of plasmids was carried out using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturers' instructions.
All plasmid purification was performed using the QIAprep spin miniprep kit (Qiagen) and all purification from agarose gels was performed using the Gel extraction kit (Qiagen), both according to the manufacturers' instructions.
All plasmids generated were sequenced to ensure there were no errors in the cloned genes (see additional file
Free full text: Click here
