Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli
© Puertas and Betton; licensee BioMed Central Ltd. 2009
Received: 09 September 2009
Accepted: 29 October 2009
Published: 29 October 2009
Despite advances in expression technologies, the efficient production of heterologous secreted proteins in Escherichia coli remains a challenge. One frequent limitation relies on their inability to be exported to the E. coli periplasm. However, recent studies have suggested that translational kinetics and signal sequences act in concert to modulate the export process.
In order to produce leech carboxypeptidase inhibitor (LCI) in the bacterial periplasm, we compared expression of the natural and optimized gene sequences, and evaluated export efficiency of LCI fused to different signal sequences. The best combination of these factors acting on translation and export was obtained when the signal sequence of DsbA was fused to an E. coli codon-optimized mature LCI sequence. When tested in high cell density cultures, the protein was primarily found in the growth medium. Under these conditions, the engineered expression system yields over 470 mg.l-1 of purified active LCI.
These results support the hypothesis that heterologous secreted proteins require proper coupling between translation and translocation for optimal high-level production in E. coli.
Escherichia coli is by far the simplest, but one of the most widely used host cell for the production of recombinant proteins . Nevertheless, the efficient translocation across the inner membrane and proper periplasmic folding of eukaryotic proteins stabilized by multiple disulfide bonds remains challenging for this organism . Unfortunately, many proteins of which there is a great biotechnological or biomedical interest are secreted proteins containing essential disulfide bonds for their native structure. Either premature cytoplasmic protein folding or incorrect disulfide bond formation in the bacterial periplasm are two known limitations in the overproduction of secreted proteins . Recently, it has been reported that signal sequences promoting co-translational translocation improved the translocation of heterologous proteins . Therefore, targeting these recombinant precursors to the cotranslational signal recognition particle (SRP) dependent pathway conceivably could result in much higher levels of periplasmic proteins than directing them posttranslationally to the SecYEG translocase . Strategies to overcome folding problems due to disulfide bond formation have primarily focused on the co-production of protein disulfide isomerases . For example, the overproduction of DsbC, a periplasmic thiol isomerase, resulted in large amounts of native human tissue plasminogen activator .
In the present study, we have investigated the production of leech carboxypeptidase inhibitor (LCI) in the periplasm of E. coli. This protein is composed of 66 amino acid residues forming a globular domain with five-stranded β-sheet and a short α-helix that are stabilized by four disulfide bonds . Like other small disulfide-rich proteins, the active conformation of LCI is strictly dependent upon the correct formation of disulfide bonds . Found in the digestive track of leeches, LCI is a strong inhibitor of human pancreatic and plasma carboxypeptidases, and thus has considerable biomedical interest . Indeed, by targeting the thrombin-activatable fibrinolysis inhibitor (TAFI) involved in hemostasis, LCI could play an important role in thrombotic disorder therapy . The binding and inhibition activity of LCI is primarily exerted by its C-terminal extremity that interacts with the active site of metallo-carboxypeptidases. In order to overproduce LCI in the periplasm, an E. coli codon-optimized sequence and different signal sequences were evaluated using a tightly controlled expression vector, suitable for high cell density cultures.
Results and Discussion
Construction of LCI precursors
MalEss and DsbAss promote high-level of LCI in the periplasm
Distribution of native LCI between cells and culture medium
Whole cell lysates
Export efficiency of preLCI
High level production of LCI in a fermentor
In this study, we found that E. coli codon optimization in the LCI gene when combined to the signal sequence of DsbA allowed the production/purification of 470 mg of active LCI per liter of culture. While codon usage may be an important criterion for translation rate [17, 18] and/or protein folding [18–20], our studies indicate that, besides the nature of signal sequence, it is also an important parameter to ensure an efficient export of heterelogous precursors. If the nature of signal sequence determines the targeting pathways , the correct combination of both parameters appears to be necessary for optimal coupling of translation to protein translocation in E. coli.
Bacterial strain and plasmids
The E. coli LMG194 strain [F- ΔlacX74 galE galK thi rpsL ΔphoA Δara714 leu::Tn10] carrying the araBAD deletion  was used as the expression host throughout the experiments. Recombinant DNA manipulations were performed as described in established protocols . Plasmid pLCB was constructed in two steps from the pBAD33 expression vector . First, the residual bla sequence was deleted by Bgl I-Tth 111I digestion and filling in with Klenow fragment. Second, a DNA fragment which contained the Shine-Dalgarno sequence comprising an ATG start codon within a Nde I site from the pIVEX2.3MCS vector  was amplified using 5'-AAGAGCTCGAATTCCATATGTATATCTCCTTGCTAGCCCAAAAAAACGGGTATGG-3' and 5'-GTAACAAAGCGGGACCAAAGCC-3' as primers, and pBAD33 as DNA template. The PCR product was digested with Mlu I and Sac I, and cloned into the same restriction sites of the previous pBAD33 derivative. The structure of the resulting plasmid was confirmed by sequencing and designated as pLCB. The mature LCI sequence was codon optimized for E. coli expression and chemically synthesized by Geneart (Regensburg, Germany). The substitution of malE or dsbA signal sequence was generated by overlap extension PCR as previously described .
For shake flask cultures, cells were grown in 100 ml of LB medium supplemented with chloramphenicol (30 μg.ml-1). Induction of the araB promoter was accomplished by addition of L-arabinose to a final concentration of 0.2%. After 6 h at 37°C, cells were harvested by centrifugation at 6,000 rpm for 15 min. For high cell density cultures, bacteria were grown in a Sartorius Biostat B® 2-L fermentor at 37°C. The aeration rate and stirrer speed were regulated to keep the dissolved oxygen concentration at 60% of its saturation value. Precultures (80 ml) were prepared in shake flasks at 37°C to mid-log phase, and then added into the fermentor containing 800 ml of the HDM medium  supplemented with chloramphenicol (30 μg.ml-1). Induction was accomplished by addition of L-arabinose (0.5%). Cell biomass was monitored by measuring both the optical density at 600 nm (OD600) and dry cell weight (DCW) as previously described . Cell viability was determined by using the LIVE/DEAD BacLight kit (Invitrogen) in combination with flow cytometry as described by the manufacturer . Plasmid stability was assessed by plating properly diluted amounts of culture samples on LB-agar plates containing 0.5% arabinose without antibiotic and with 30 μg.ml-1 chloramphenicol. After overnight growth at 37°C the numbers of colony forming unit (CFU) were determined.
Cell fractionation and protein assays
Cells carrying the pLCB derivatives, normalized to the same OD600, were fractionated by spheroplast preparation as previously described . To analyse secreted LCI in the culture media, culture supernatants were applied to SepPak Plus C18 cartridges (Waters) pre-equilibrated by 10% acetonitrile. Then, the columns were washed with 10% acetonitrile, and proteins were eluted by 30% isopropanol. Total protein content was determined by the Bradford assay using bovine serum albumin as a standard. Cellular fractions were separated on 10% Bis-Tris polyacrylamide NuPage gels (Invitrogen), and proteins were visualized by Coomassie blue staining. For quantitative analysis, gels were scanned with Gel Doc XR imaging system (Biorad).
After cultivation, cells were centrifuged as described above and supernatants were filtered through a 0.22 μm syringe filter (Millipore). LCI was purified by reverse phase chromatography using a Ultimate 300 HPLC system (Dionex) and a Vydak C4 column, with a linear gradient ranging from 20 to 80% acetonitrile at a flow rate of 1 ml.min-1 as previously described . To quantify the concentration of native LCI found in periplasmic and culture supernatants, a calibration curve was constructed by using purified active protein as a standard. The LCI activity was assayed using the Carboxypeptidase A assay kit (Sigma Aldrich) in 50 mM Tris-HCl buffer, pH 7.5; containing 100 mM NaCl.
We thank FX Avilès and his collaborators for many helpful discussions, and E. Johnson for critical reading of the manuscript. JM Puertas is a recipient of the Spanish Ministry of Science and Innovation (MICINN). This work was supported in part by the Institut Pasteur and the Centre National de la Recherche Scientifique (CNRS), and by a grant from the Agence Nationale de la Recherche (O6-BLAN-023904).
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