Protein expression constructs

In the present study, recombinant bacterial collagen, VCL, has been expressed using the pCold vector system, as it had previously been shown that this system was effective in shaker culture for production of the protein in the E .coli host strain BL21[18, 25]. The construct was termed VCL, where V is the non-triple-helical registration domain and CL is the triple-helical, collagen-like domain (Figure 1). The pCold vectors selectively induce target protein synthesis at low temperatures where the synthesis of host proteins is reduced or suppressed and protease activity is decreased. The pCold system is designed for efficient protein expression utilising a promoter derived from the cspA gene, which is a cold-shock gene, with a lac operator inserted downstream to control expression[26]. In addition, expression of a second construct, VCLH, which included a heparin binding domain, H, substituted using site directed mutagenesis into the CL sequence, was also tested. In this construct, a heparin binding sequence, GRPGKRGKQGQK, derived from the collagenous tail of acetylcholine esterase[23, 24] was substituted via PCR directed integration into the CL sequence GPAGPMGPAGER that starts at base pair 564/amino acid residue 188 (Figure 1). The substitution was confirmed by DNA sequencing. The pCold vector system is available as a research tool; it is possible that proprietary commercial vectors could give much better yields than observed in the present study that has used the pCold system. Recombinant proteins, VCL and VCLH, were isolated in sufficient quantities for use as electrophoretic quantitation standards (Figure 2A) using a conventional purification protocol (Ni metal affinity column chromatography and with binding via a His₆ tag incorporated at the N-terminus of the protein). Final purification was achieved by gel permeation chromatography[25].

Protein expression in shake flasks

The expression of VCL in shake flasks using 2xYT Media and Defined Media (DM) were used as the baseline process for this study. Cells were grown for 24 h at 37°C, after which an OD_{600 nm} of around 6 was achieved, prior to induction at 25°C. Expressed protein production was determined after incubation at 25°C for 10 h, followed by a further 14 h at 15°C. It was hoped that the additional reduction in temperature would maintain the selective target protein production achieved by the pCold vector system. The changes in cell densities were tracked throughout the process (Figure 3A), and at the end of the production phase, moderate amounts of wet cell paste, 8.3 g/L for DM and 9.7 g/L for 2xYT, were obtained. Expression yields were determined by comparison to known amounts of VCL protein by SDS-PAGE (Figure 2A). These shake flask processes achieved recombinant collagen expression levels of around 0.2 g/L in DM and 0.3 g/L in 2xYT (Table 1). These yields are adequate for biochemical and biophysical studies of VCL and related constructs such as VCLH, but are well below production levels required for commercial production.

Protein expression in 2 L stirred tanks – batch process (low cell density)

Expression of both the VCL and the VCLH was tested using a batch process in a 2 L stirred tank bioreactor. After inoculation, both processes were maintained at 37°C for the first 5 hours to increase the cell density prior to induction. After this initial period, the culture had an optical density OD_{600 nm} in the range of 2 to 3. The cultures were then cooled to 25°C, induced with IPTG and incubated for a further 7 h. For both constructs, wet cell paste yields were between 5 and 6 g/L. The increase in cell density was tracked throughout (Figure 3A), showing, steady increases in both cases. The final optical densities were below those achieved in shake flasks reflecting the shorter times for both the growth and induction phases in the bioreactor experiments. Despite the shorter incubation times, both trials led to moderate recombinant collagen yields, 0.7 to 1.0 g/L. Transfer of the process to a more controlled bioreactor resulted in a 3-fold increase in total collagen production and a 4-fold increase in specific productivity (mass of collagen per cell), although further yield increases are required to meet the requirements of commercial production.

Protein expression in 2L stirred tanks – fed-batch process (high cell density)

Both the VCL and VCLH constructs were used for initial testing of expression using a high cell density, fed-batch process. A high cell density process was implemented to increase collagen production. If the specific collagen productivity (mass of collagen per cell) can be maintained then an increase in cell density will result in an increase in collagen production. To achieve a high cell density prior to induction, the initial growth phase was extended to 24 h during which cell densities typically exceeding OD_{600 nm} of 80 were obtained. Again, the temperature was reduced to 25°C and recombinant protein expression induced by the addition of IPTG; the culture was incubated for a further 10 h. The process resulted in a significant improvement in collagen yields; 8.2 g/L for VCLH and 9.5 g/L for VCL, indicating that this fermentation approach has potential for high level production of the recombinant protein. The specific productivity was less compared to the low cell density process but due to the large increase in cell mass the volumetric productivity increased almost 10-fold. As the differences between VCL and VCLH production were relatively small, only VCL was used for further process optimisation.

The previous experiment showed that a 12°C temperature decrease resulted in collagen yields in excess of 8 g/L. The effect of induction temperature was examined further by following a similar protocol, except the induction was changed from 25°C to 15°C and the induction time was increased from 10 to 24 h. This process was used to test whether a lower temperature would further reduce host cell protein production resulting in greater enrichment of recombinant collagen. The lower induction temperature did result in greater enrichment of the target protein (the specific productivity increased 2-fold) whilst volumetric production was similar to the previous experiment (9.5 g/L at 25°C and 10 g/L at 15°C). The final optical density was lower at 15°C (OD_{600 nm}= 65) compared to 25°C (OD_{600 nm}= 88) despite the induction time being increased from 10 to 24 hours. The unchanged volumetric production was most likely the result of slower cell growth at the lower temperature as shown in Figure 3B; cell growth is negligible after reducing the temperature to 15°C.

To further increase recombinant collagen production the extended induction time was retained and stepwise temperature reduction was introduced into the process to enhance pCold’s “cold shock selectivity”. After induction for 10 h at 25°C, the temperature was reduced to 20°C and the culture held at this temperature for 5 h. The temperature was then reduced again to 15°C and held at this temperature for a further 9 h. This process change resulted in a further marked yield increase with recombinant collagen production levels of 13.0, 19.0 and 19.3 g/L in three separate batches (average of 17.1 g/L) – production levels that are potentially within a commercially feasible range. The stepwise temperature reduction resulted in an increase in final optical density without too greater impact on specific productivity. Recombinant collagen production was followed during the induction phase of the process using SDS-PAGE (Figure 2B) which shows a steady increase in VCL production during the initial hours after induction with a further increase at the end of the process. In addition, purified protein samples from cell aliquots, for use as gel standards, also showed that the collagen domains of the expressed products were resistant to trypsin digestion[19] indicating that the collagen domains had folded into the triple-helical conformation which confers proteolytic resistance[19].

In the present study, we have shown that good production levels of VCL can be achieved, with a maximum production of 19.3 g/L. After removal of the His₆ tag and the V-domain, which would probably be necessary for biomedical applications of the collagen-like domain, this equates to a maximum yield of the CL domain of about 14.1 g/L, with a further loss of yield depending on the efficiency of any purification protocol. The use of the His₆ tag is efficient for laboratory purifications[19, 25], but would not be suitable for large scale commercial preparations. Other affinity approaches may be more useful and cost effective, including the use of specific collagen binding proteins[27]. The present study has shown that excellent recombinant collagen yields can be obtained using a high cell density fed-batch process using pCold vectors. The best yields obtained compare favourably with the average yields reported for other bacterial expression studies of 14 g/L[28], although there may be higher commercial yields that remain commercial-in-confidence. Nevertheless, use of alternative vector systems that may be better suited for use at high cell densities may lead to even further increases in yield. The present yield, however, compares favourably with fed-batch fermentation yields obtained using P. pastoris and Hansenula polymorpha. These recombinant systems were used to produce a 30 kDa fragment containing an N-propeptide which after cleavage gives a 14 kDa mammalian collagen fragment in the yeasts P. pastoris and Hansenula polymorpha (recently designated as a Pichia species)[10]. These data gave the best yield with H. polymorpha expression of around 0.6 g/L, well below the bacterial collagen yields achieved in this study. Nevertheless, much better yields, better than 2 g/L, could be obtained from P. pastoris when used in commercially optimised production[12].

Recombinant construct

The fragment of S. pyogenes Scl2.28 gene[15] encoding the globular (V) and the collagen-like (CL) domains, and the variations to include a His-tag at the N-terminal of the V-domain and an enzyme cleavage site between the V- and CL domains, was cloned into the pColdIII vector (Takara Bio, Shiga, Japan), giving construct VCL, and transformed for expression in E. coli strain BL21 as previously described[25]. In addition, the construct was cloned into the shuttle vector pSL1180 using restriction sites 5^′ NdeI and 3^′ BamHI. This clone was used for site directed mutagenesis to introduce a heparin binding sequence[23, 24] GRPGKRGKQGQK at base pair 564 via PCR directed integration, giving construct VCLH. PCR products were treated with DpnI, to digest all parental DNA and subsequently transformed into E. coli strain XL1-BLUE. Selected DNA clones were sequenced and those containing the required heparin binding domain was subcloned into pColdIII vector (Takara Bio, Shiga, Japan) using the 5^′ NdeI and 3^′ BamHI sites. Positive clones were selected and transformed into E. coli host BL21 for expression. Schematics of the two constructs are shown in Figure 1. Prior to expression studies, all constructs were confirmed by DNA sequencing.

Media

Complex medium (2xYT) contained 16 g tryptone, 10 g yeast extract and 5 g NaCl per litre. The defined medium (DM) used in this study contained per litre: KH₂PO₄, 10.6 g; (NH₄)₂HPO₄, 4.0 g; citric acid, 1.7 g; glucose, 25 g; MgSO₄.7H₂O, 1.23 g; ampicillin, 200 mg; thiamine hydrochloride, 4.4 mg; and trace salts solution 5 mL. The trace salts solution contained per litre: CuSO₄.5H₂O, 2.0 g; NaI, 0.08 g; MnSO₄.H₂O, 3.0 g; Na₂MoO₄.2H₂O, 0.2 g; boric acid, 0.02 g; CoCl₂.6H₂O, 0.5 g; ZnCl₂, 7.0 g; FeSO₄.7H₂O, 22.0 g; CaSO₄.2H₂O, 0.5 g and H₂SO₄, 1 mL. As required, glucose, magnesium, trace salts, thiamine and ampicillin were aseptically added as concentrated stock solutions to the media after sterilisation.

Seed cultures

Primary seed cultures were prepared from single colonies taken from a fresh transformation plate, and grown in 10 mL of 2xYT (in a 30 mL bottle) containing 10 g/L glucose and 200 μg/mL ampicillin. The culture was incubated at 37°C shaking at 200 rpm for 24 h. A volume (0.5 mL) of the primary seed culture was used to inoculate 500 mL of 2xYT (in a 2L Erlenmeyer flask) containing 10 g/L glucose and 200 μg/mL ampicillin. This secondary seed culture was incubated at 37°C shaking at 200 rpm for 16 h.

Protein expression in shake flasks

Cultures were grown in 2 L Erlenmeyer flasks containing 500 mL of either 2xYT or DM media and incubated at 37°C for 24 h. The cultures were then cooled to 25°C and protein expression induced by addition of 0.5 mM isopropyl beta-D-thiogalactopyranoside (IPTG). The cultures were then incubated at 25°C for 10 h and for a further 14 h at 15°C. Samples were taken during the course of the incubation and the cells pelleted by centrifugation followed by disruption using Bugbuster™ (Merck) and quantification by SDS-PAGE (as described below).

Protein expression in 2L stirred tanks

Recombinant bacterial collagens were produced in 2 L stirred tank bioreactors connected to a Biostat B (Sartorius Stedim Germany) control system. The initial volume of medium in the fermenter was 1.6 L and glucose as used as the carbon source. A volume of the secondary seed culture was added to the bioreactor to attain an initial optical density (measured at 600 nm) of 0.25. Foaming was controlled via the automatic addition of 10% (v/v) polypropylene glycol 2025; 3 mL of the antifoam solution was added prior to inoculation. The pH setpoint was 7.0, controlled by automatic addition of either 10% (v/v) H₃PO₄ or 10% (v/v) NH₃ solutions. The dissolved oxygen setpoint was 20% of saturation and a two-step cascade control was used maintain the dissolved oxygen above the specified setpoint. The agitator speed ranged from 500 rpm to 1200 rpm and airflow (supplemented with 5% pure O₂) ranged from 0.3 L min^-1 to 1.5 L min^-1. For the high cell density fed-batch processes, the feed solution was comprised of 400 mL of 660 g/L glucose solution to which 40 mL of 1 M MgSO₄7H₂O added. The feed flow rate was 15 mL h^-1 and the feed was initiated 8.5 h after inoculation. Incubation times and temperatures for individual experiments are given in Table 1. The culture was cooled (over a 20 minute period) to the required temperature 24 h after inoculation to activate the cold shock component of the vector and protein expression induced by addition of 1 mM (final concentration in the culture) IPTG.

Preparation of protein standards

Recombinant VCL and VCLH protein standards were extracted from wet cell paste by sonication in 40 mM sodium phosphate buffer, pH8.0, 1 mM phenylmethanesulfonyl fluoride, using 20 ml buffer per gram of cell paste. After centrifugation, clarified supernatant was taken to 20 mM sodium phosphate 300 mM NaCl and 30 mM imidazole buffer, pH8.5, and absorbed onto a Ni charged HyperCel-Sepharose metal ion affinity resin (Pall Life Sciences). Elution was by the same buffer, but containing 500 mM imidazole. Eluted fractions containing recombinant protein were pooled, concentrated and exchanged into 20 mM sodium phosphate buffer, pH8.0, using a 10 kDa cross-flow filtration membrane (Pall Life Sciences), followed by further purification in the same buffer on a Sephacryl S200 26/60 column (GE Healthcare). After additional treatment of purified VCL and VCLH with trypsin[19], SDS-PAGE[29] as described for Sample Analysis, was used to show that the triple helix remained intact.

Sample analyses

Samples were taken from the cultures throughout each process. Cell mass was determined by measuring the optical density of diluted samples at 600 nm (OD_{600 nm}). VCL or VCLH were quantified in small sub-samples and to correct for changes in cell numbers, the formula 200/OD_{600 nm} was used to determine the volume of culture required for SDS PAGE analysis. The aliquot was centrifuged and the cell pellet lysed by addition of 40 μL of Bugbuster™ (Merck) and incubated for 1 h. After the incubation, 10 μL 5x sample buffer containing 5% (v/v) 2-mercaptoethanol was added to the lysate and the mixture heated at 95°C for 1 min prior to electrophoresis. SDS PAGE was completed as described by[29] using NuPAGE (Invitrogen) 4-12% Bis-Tris gels with MES running gel buffer, at 180 V for 60 min, followed by staining with Coomassie Blue R-250. After destaining, the band corresponding to VCL or VCLH bands were quantified by densitometric analysis using Multi Gauge V3.0 FujiFilm software and compared to known standard loadings of purified VCL protein.