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, 30, 31]. 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 2A). 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 file 2 : Table of vectors used in this study for plasmid names and details).
For expression in LB media, E. coli strains containing expression vectors were streaked out from glycerol stocks stored at -70°C onto LB agar plates containing suitable antibiotics to allow for selection (100 μg/ml ampicillin for pET23 derivatives, 35 μg/ml chloramphenicol for pLysS derivatives; with 10 μg/ml tetracycline and 15 μg/ml kanamycin for selection of origami or rosetta-gami strains). The next day one colony from these plates were used to inoculate 5 ml of LB media, containing suitable antibiotics, and grown overnight at 30°C, 200 rpm. This overnight culture was used to seed a 50 ml culture of LB containing suitable antibiotics in a 250 ml conical flask to an optical density of 0.05 at 600 nm (OD600). The addition of FAD to the media is not required for the production of active Erv1p and preliminary experiments suggestion such addition does not increase the yield of active proteins generated in this system. This culture was grown at 30°C, 200 rpm until the OD600 reached 0.4 at which point protein production was induced either by the addition of 0.5 mM IPTG or for pre-induction of the folding factors by the addition of 0.5% w/v arabinose followed 30 minutes later by 0.5 mM IPTG. The cells were then grown for a total of 4 hours post induction at 30°C, 200 rpm and the final OD600 measured. The cells were collected by centrifugation and resuspended to an OD600 equivalent of 10 (based on the final OD600 of the culture) in 20 mM sodium phosphate pH 7.4, 20 μg/ml DNase, 0.1 mg/ml egg white lysozyme and frozen. Such normalization allows for easy correction for differences in the growth rates of the cultures and allows rapid equal total protein loading of samples for activity assay and SDS-PAGE analysis. Cells were lysed by freeze-thawing. Where appropriate, the insoluble fraction was removed by centrifugation and the soluble fraction removed quickly to a new container. Cell lysates or soluble fractions were stored frozen in 1 ml aliquots for further experiments as repeated freeze-thawing clearly influenced the results obtained in a protein dependent manner.
For expression in EnBase media (Biosilta Oy, Finland) E. coli strains containing expression vectors were streaked out from glycerol stocks stored at -70°C onto LB agar plates containing suitable antibiotics to allow for selection. The next day three loopfuls of bacteria from the region of single colonies were collected and resuspended in 0.8 ml of EnBase media. This was used to inoculate 25 ml of EnBase media (with thiamine, magnesium and Enz I'm pre-added according to the manufacturer's instructions), containing suitable antibiotics in a 250 ml conical flask to an OD600 of 0.1. The flask was then sealed with an oxygen permeable seal and the bacteria grown for 15 hours at 30°C, 200 rpm. The OD600 of the culture was measured to ensure correct growth and the booster media added according to the manufacturer's instructions. Pre-induction of the folding factors was performed by the addition of 0.5% w/v arabinose, the cells grown for 30 minutes and then the protein of interest induced with 0.5 mM IPTG and the cultures grown for a further 23.5 hours and the final OD600 measured. The post culture processing was as per growth in LB media.
Note, in addition to the results shown here many of the proteins of interest were also expressed under conditions other than those listed above. Varying the conditions altered the yields of active protein obtained, sometimes in a protein dependent manner. However, all of the results obtained from all conditions are consistent with the results reported here. While these consensus conditions may not be optimal for any individual protein in our system, they appear to be a good starting point for screening for any protein in our system. Note also that 30°C for growth was chosen as Erv1p is not well expressed in an active form at 37°C and that growth of large numbers of cultures was more convenient for us at 30°C than at lower temperatures. Some proteins, for example the luminal domain of tissue factor (data not shown), are expressed more homogeneously at lower temperatures, but the effect of temperature, induction OD600, induction time or the concentration of inducing agent has not been systematically tested for any protein.
vtPA activity measurements from lysates
vtPA activity was measured using a chromagenic substrate, chromozyme t-PA peptide assay (Roche), using a methodology similar that that recommended by the manufacturer but adapted for a continuous assay in 96-well plate format. Since this method showed slight variations in activity with time all of the vtPA activity measurements were repeated using the same batch of substrate and same buffers and that data is presented here. 20 mg of substrate was dissolved in 4 ml of sterilized water to generate a 20× substrate stock solution. 10 μl of soluble fraction from cell lysates were added to 190 μl of substrate diluted in reaction buffer (100 mM tris-HCl, 0.15% tween20; pH 8.5) to give a final concentration that is 1× in a 96 well microtitre plate, thermally equilibrated to 37°C. The absorbance at 405 nm was measured at 3 minute intervals for 30 minutes to determine the rate of formation of the product. Background rates from appropriate controls strains not expressing vtPA were subtracted, with these being less than 5% of the signals for any system where Erv1p and a disulfide isomerase were co- or pre-expressed. For samples with little vtPA activity 20 μl of soluble fraction from cell lysates were added to 180 μl of 1× substrate (see additional file 1 : Table of vtPA production and activity data). Control samples tested with 10 and 20 μl of lysate showed no significant differences in activity once normalized for the volume of lysate used. It should be noted that this assay format gives a very inaccurate measure for the production of vtPA with no co- or pre-expression of vtPA in a wild-type E. coli background. Furthermore, our vtPA constructs have different tags than previously reported and our activity measurements are made from E. coli lysates, both of which may affect activity measurements and hence cross-comparisons to available specific activity values are inappropriate. Hence all of the data has been normalized to that produced with co-expression of DsbC in a Δ gor Δ trxB background i.e. the best system previously reported. More accurate determinations of very low activities can be obtained under different experimental conditions (more lysate and much longer incubations), but Erv1p co- or pre-expression strains cannot be assayed under these conditions as the measurements go off scale. All samples were measured at least in duplicate. The number of samples (n) in Figures 1, 3 and 4 and Table 1 represent independent bacterial cultures, each measured at least in duplicate, implying the number of activity measurements for each data point varied from 4 to 24.
Protein purification and analysis
Purification of hexa-histidine tagged proteins was performed by standard immobilized metal affinity chromatography using Ni-NTA columns (Qiagen) under native conditions was performed according to the manufacturers' instructions following clearance of the cell lysate by centrifugation (10000 rpm, 15 minutes, 4°C).
Immobilized metal affinity chromatography of N-terminal hexa-histidine tagged BPTI wild-type and mutants was performed using a 5 ml HiTrap Chelating HP column (GE healthcare). The soluble fraction of the E. coli lysate was prepared by centrifugation (10000 rpm, 15 minutes, 4°C). This was then loaded onto the HiTrap Chelating column which had been pre-charged with Ni2+, washed and equilibrated with 3 column volumes of 20 mM sodium phosphate (pH 7.4). After loading the sample the column was washed with 3 column volumes of wash buffer (20 mM sodium phosphate, 50 mM imidazole, 0.5 M sodium chloride; pH 7.4) then 3 column volumes of 20 mM sodium phosphate (pH 7.4) before elution with 2 column volumes of 50 mM EDTA (pH 7.4). All buffers were filtered and degassed before use.
Reverse phase high pressure liquid chromatography (rpHPLC) analysis of BPTI wild type and mutants produced in our strains was performed on an ÄKTA FPLC system (GE Healthcare) using a μRPC C2/C18 ST 4.6/100 column: The column was pre-equilibrated in buffer A (0.065% trifluoroacetic acid), before a 50 μl sample was loaded to the system using automatic sample injection. The elution gradients used were: 20-30% buffer B (90% acetonitrile, 0.05% trifluoracetic acid) over 3 column volumes followed by a column volume of 30% buffer B, then 30-31% buffer B over 5 column volumes, 31-45% buffer B over 5 columns volumes, 1 column volume of 45% buffer B for 1 column volume and then 45-100% buffer B over 3 column volumes. 0.5 ml fractions were collected with a flow rate of 0.3 ml/min. All buffers were filtered and degassed before use.
Purification of MBP-tagged proteins was performed using amylose resin (New England Biolabs). Amylose resin (500 μl for 5 ml of lysate) was packed into an empty plastic column. The resin was washed twice with 2 ml of water and then equilibrated with 5 ml of 20 mM sodium phosphate buffer (pH 7.4). The soluble fraction of the E. coli lysate was loaded on the column and allowed to equilibrate at room temperature for 5 minutes. The column was then washed five times with 2 ml of wash buffer (20 mM sodium phosphate, 0.2 M sodium chloride pH 7.4). 150 μl of elution buffer (20 mM sodium phosphate, 10 mM maltose, 0.2 M sodium chloride, pH 7.4) was added and the column allowed to equilibrate at room temperature for 5 minutes. The elution was repeated two more times.
For Factor Xa cleavage 5 μl of 10× Xa-buffer (New England Biolabs) was added to 43 μl of protein solution (in the MBP-purification elution buffer) with a final amount of protein of circa 50 μg. 2 μl of 1 mg/ml Factor Xa (New England Biolabs) was added, mixed and the solution incubated for 6 hours at room temperature. The soluble and insoluble fractions were subsequently analysed by reducing and non-reducing SDS-PAGE.
Elman's assay for free thiol content were performed at room temperature under denaturing conditions in 50 mM Tris buffer, 2 M Guanidine hydrochloride (pH 8.0) using 0.073 mg/ml Elman's reagent. The change in absorbance at 412 nm was monitored after 15 minutes and the free thiol content calculated using a molar extinction coefficient of 13600 M-1cm-1.