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Heterologously expressed proteins in Escherichia coli may undergo unwanted N-terminal processing by methionine and proline aminopeptidases. To overcome this problem, we present a system where the gene of interest is cloned as a fusion to a self-splicing mini-intein. Furthermore, this fusion construct is expressed in an engineered Escherichia coli strain from which the pepP gene coding for aminopeptidase P has been deleted. We describe a protocol using human cationic trypsinogen as an example to demonstrate that recombinant proteins produced in this expression system contain homogeneous, unprocessed N-termini.
The expression system presented here was developed as part of an effort to elucidate the functional effect of the p.A16V mutation in human cationic trypsinogen, which has been reported to be associated with chronic pancreatitis by several studies (1–4). This variant alters the N-terminal amino acid residue of the mature, secreted trypsinogen protein. The amino-acid numbering starts with Met1 of the pre-trypsinogen protein and the first 15 residues comprise the secretory signal peptide. It had previously been determined that other pancreatitis-associated mutations in human cationic trypsinogen increase the propensity of trypsinogen for autoactivation (5,6). We speculated that the p.A16V mutation might have a similar effect; however, functional characterization of the recombinantly expressed mutant trypsinogen required preparations with uniform, authentic N-termini.
For high-yield heterologous expression of human cationic trypsinogen in Escherichia coli, an expression plasmid was constructed in which the secretory signal peptide of trypsinogen was deleted and the initiator methionine was placed immediately upstream of the mature protein. Trypsinogen expressed from this construct accumulated in the cytoplasm as inclusion bodies. The native N-terminal sequence of trypsinogen isolated from pancreatic juice is Ala16-Pro17-Phe18. The expected N-terminal sequence of trypsinogen expressed in E. coli is Met- Ala16-Pro17-Phe18. However, we found that the N-terminal sequence of recombinant trypsinogen in the inclusion bodies was heterogeneous, consisting of ~30% Pro17-Phe18 and approximately ~70% Met-Ala16-Pro17-Phe18. Apparently, part of the expressed trypsinogen was processed by methionine aminopeptidase and then by proline aminopeptidase (aminopeptidase P). Removal of the initiator methionine by methionine aminopeptidase is a well documented phenomenon in E. coli (7) which occurs if the second amino acid residue has a small, uncharged side chain. With high-level expression of heterologous proteins, the enzyme gets saturated and only a fraction of the proteins is processed. After cleavage of the initiator methionine, proteins can be subject to cleavage by aminopeptidase P, which cleaves the N-terminal amino acid of a protein if proline is in the penultimate position (8).
To address the problem of unwanted N-terminal processing by aminopeptidases, we developed a novel expression system (9). First, the gene of interest (in this case human cationic trypsinogen) was cloned in a C-terminal fusion with the Synechocystis DnaB mini-intein (10). In this fusion construct (see Fig. 1.), translation was initiated by the start codon of the intein gene and the intein moiety was subsequently removed through intein self-cleavage (11). To eliminate cleavage by aminopeptidase P, the fusion construct was expressed in the aminopeptidase P deficient E. coli LG-3 strain. This strain was engineered by deleting the pepP gene coding for aminopeptidase P from the E. coli chromosome (12, 13), using the recombination-based method described by Datsenko and Wanner (14). The intein-trypsinogen fusion was expressed in LG-3 cells as inclusion bodies, solubilized with guanidine and re-natured in vitro followed by affinity purification on immobilized ecotin (15). Finally, we used MonoS cation-exchange chromatography to remove the small fraction of uncleaved intein fusion proteins and to obtain a pure trypsinogen preparation with uniform, authentic N-termini. The expression system described here can be useful for the heterologous expression of proteins whose N-terminal integrity is compromised by aminopeptidase activity.
To remove the pepP gene that encodes an active proline aminopeptidase from the E. coli genome, the Red recombinase based gene deletion system described by Datsenko and Wanner (14) is used. First, a recombination substrate is produced by amplifying the kanamycin resistance gene flanked by two FRT sites using primers that contain 44–45 nucleotide extensions homologous to sequences flanking the pepP gene on the E. coli chromosome. This PCR product is introduced into E. coli cells harboring a helper plasmid expressing the λ Red recombinase that catalyzes homologous recombination between the recombination substrate and the E. coli chromosome. The target gene is thus exchanged for the recombination substrate containing the kanamycin resistance gene and recombinants can be selected for by kanamycin resistance. The kanamycin resistance gene is eliminated by site-specific recombination between the two FRT sites catalyzed by the FLP recombinase introduced on a second helper plasmid. Both helper plasmids are temperature sensitive and can be eliminated by growing at the non-permissive temperature. At the end of the procedure, a single FRT site remains at the locus of the pepP gene. This remaining FRT sequence is sometimes termed a “scar sequence”.
Ecotin is a pan-serine-protease inhibitor from E. coli (18) that forms a relatively tight complex with trypsinogen and thus can be used for affinity purification of the zymogen (15). Recombinant ecotin (17) is overexpressed in the periplasmic space of E. coli and isolated using osmotic shock (19) followed by trypsin affinity chromatography. Purified ecotin is immobilized on aldehyde activated resin by reductive amination using cyanoborohydride (15) and loaded into a chromatography column.
Trypsinogen variants cloned into the intein fusion constructs are expressed as inclusion bodies in E. coli LG-3. Analysis of solubilized inclusion bodies by SDS-PAGE (see Subheading 3.3.4) reveal that the intein moiety has been cleaved off approximately 70–80% of the fusion proteins in E. coli (Fig. 2A), therefore a separate in vitro intein cleavage step is not included. After in vitro refolding, trypsinogens are purified using ecotin affinity chromatography (15). SDS-PAGE analysis of purified samples show that eluted trypsinogens contain a small fraction of uncleaved fusion proteins (Fig. 2B) and further purification is necessary. The eluate is thus subjected to ion-exchange chromatography (Fig. 2D) which yields a homogenous preparation of recombinant trypsinogens with authentic N-termini (Fig. 2C).
We would like to thank Barry L. Wanner (Department of Biological Sciences, Purdue University, West Lafayette, IN) for sharing the plasmids and bacterial strains of the Red recombinase based gene deletion system. The valuable support and contributions of Ronald Kaback, Edit Szepessy, Zoltán Kukor and Miklós Tóth are gratefully acknowledged. This work was supported by NIH Grant DK058088 to M.S.-T.
1There are several newer generation forms of Actigel ALD available (e.g. Ultraflow, Superflow) which claim to exhibit better flow rates. In our application we experienced no benefit from using these resins and we obtained the best results with the least expensive, regular Actigel ALD resin.
2Add dithiothreitol immediately before use from a 1 M solution kept at –20°C in aliquots.
3Solutions containing methanol are hazardous and should be disposed of accordingly.
4Gel-Dry™ solution contains ethanol, polyethylene glycol, methanol and isopropanol and should be disposed of as hazardous waste.
5The underlined sequences in primers B and C overlap and thus allow the hybridization of PCR products obtained in the first round of amplifications, (see ref 20).
6In both primer sets, one primer anneals within the kanamycin resistance gene and the other anneals to a nearby sequence in the E. coli chromosome. Amplification of PCR products indicates that recombination between the pepP gene and the recombination substrate has occurred.
7Colonies resistant to kanamycin have undergone homologous recombination and have lost the pepP gene, while resistance to ampicillin indicates that the helper plasmid has not been lost.
8The coupling buffer must be free of amines, therefore, phosphate-based buffers are recommended. Do not use Tris buffer or other amine-containing buffers.
9Growing the bacterial culture followed by the isolation of the periplasmic fraction is a full-day procedure, so it is practical to start the culture as early as possible.
10At this step, the supernatant has to be clear. Centrifuge again if necessary before proceeding to step 9.
11The procedure can be paused here and the supernatant stored on ice overnight.
12Distribute the dialyzed sample to 50 mL Falcon tubes (about 15 mL in each). Using adequate cold protection, hold the tubes tilted in a Dewar flask with liquid nitrogen and slowly rotate the tubes so that ecotin freezes onto the tube wall as a layer. Cover the tubes with Parafilm and punch holes in the Parafilm to let water evaporate. Place tubes in a larger freeze-dry bottle or flask and lyophilize overnight.
14It is useful to check expression levels before starting the purification procedure. After approximately 4 hours of culturing, a 1 mL sample is removed from each culture and centrifuged for 5 min at 12,300 rpm. Discard the supernatant and resuspend the pellet in 1 mL resuspension buffer and sonicate three times for 20 s (continuous mode, power setting 5). Microfuge the samples for 5 min at 12,300 rpm and discard the supernatants. Resuspend the pellets in 30 μL reducing Laemmli sample buffer, vortex briefly and incubate at 90°C for 5 minutes. Apply these samples to a SDS polyacrylamide gel (see Subheading 3.3.4). Purified trypsinogen should be loaded as positive control and samples with a strong band at the same position are processed further.
15Pellets can be combined to produce a single pellet at the end of the washing procedure.
16First, add L-cystine in powder form to 50 mL refolding buffer and dissolve by vigorous stirring at room temperature on a magnetic stirrer for about 30 minutes. There is no need for argon at this step. L-cystine is poorly soluble in water and undissolved crystals may still be visible after 30 min. This has no impact on the refolding procedure. Add L-cysteine after the refolding solution has been equilibrated with argon for a few minutes. L-cysteine should dissolve readily.
17A 200 μL pipet tip is attached to the tubing from the gas cylinder and punched through the Parafilm covering the flask with the refolding buffer. The flask is placed on a magnetic stirrer and after a few minutes of argon flow, the denatured trypsinogen sample is added dropwise to the buffer under moderate stirring. The solution is then stirred for another 5 min under argon.
18This step is included to dilute the guanidine content of the sample. Centrifugation is necessary to remove any precipitated proteins, which can clog up the chromatography system and the column.
19Gels can be stored for two weeks at 4°C wrapped in wet paper towels in airtight plastic bags.
20To minimize exposure to methanol vapor, cover the dishes tightly with aluminum foil during these steps. Solutions containing methanol are hazardous and should be disposed of accordingly. To reduce the amount of hazardous waste, slightly stained destaining solutions can be re-used at the beginning of a destaining procedure. Speed and efficiency of destaining can be increased by placing a small sheet of Kimwipe in the dish which adsorbs colloidal stain particles.
21Care should be taken to avoid air bubbles between the cellophane sheets as these can result in white spots on the dried gels. The Gel-Dry™ solution contains ethanol, polyethylene glycol, methanol and isopropanol and should be covered during soaking of gels and disposed of as hazardous waste.