Precise and efficient cleavage of recombinant fusion proteins using mammalian aspartic proteases

Blanka Kühnel1, Joenel Alcantara2, Joseph Boothe1, Gijs van Rooijen3 and Maurice Moloney1,2,4

1SemBioSys Genetics Inc., 110 2985 23rd Avenue NE, Calgary AB T1Y 7L3, 2Department of Biosciences, University of Calgary, 2500 University Drive NW, Calgary AB T2N 1N4 and 3GenomePrairie, 115 3553 31st Street NW, Calgary AB T2L 2K7, Canada

4 To whom correspondence should be addressed. e-mail: moloneym{at}sembiosys.com


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Expression of recombinant proteins as translational fusions is commonly employed to enhance stability, increase solubility and facilitate purification of the desired protein. In general, such fusion proteins must be cleaved to release the mature protein in its native form. The usefulness of the procedure depends on the efficiency and precision of cleavage and its cost per unit activity. We report here the development of a general procedure for precise and highly efficient cleavage of recombinant fusion proteins using the protease chymosin. DNA encoding a modified pro-peptide from bovine chymosin was fused upstream of hirudin, carp growth hormone, thioredoxin and cystatin coding sequences and expressed in a bacterial Escherichia coli host. Each of the resulting fusion proteins was efficiently cleaved at the junction between the pro-peptide and the desired protein by the addition of chymosin, as determined by activity, N-terminal sequencing and mass spectrometry of the recovered protein. The system was tested further by cleavage of two fusion proteins, cystatin and thioredoxin, sequestered on oilbody particles obtained from transgenic Arabidopsis seeds. Even when the fusion protein was sequestered and immobilized on oilbodies, precise and efficient cleavage was obtained. The precision, efficiency and low cost of this procedure suggest that it could be used in larger scale manufacturing of recombinant proteins which benefit from expression as fusions in their host organism.

Keywords: chymosin/fusion protein/oilbody/oleosin/protein cleavage/recombinant expression


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The production of recombinant proteins as fusion proteins is widely used on a laboratory scale where it provides two major advantages. First, expression of the desired protein as a fusion with an affinity tag may greatly facilitate purification of the fusion by affinity chromatography (Ford et al., 1991Go). Secondly, incorporation of a highly stable protein at the N-terminus of a fusion may in some cases, increase the accumulation of the fusion protein and increase its solubility (Georgiou and Valax, 1996Go). In order to recover a recombinant protein that has been expressed as a fusion in its native form, cleavage of the fusion is required. Chemical or enzymatic cleavage of a specific peptide bond can be employed (Coligan et al., 2001Go). Chemical cleavage often requires elevated temperatures and toxic compounds, which denature the target protein and complicate downstream purification. Enzymatic cleavage is a milder procedure that in general does not denature the protein (Coligan et al., 2001Go).

For the production of pharmaceutical proteins, the authenticity of the polypeptide chain is essential. This limits the number of available cleavage agents to those that can accommodate a variety of residues on the C-terminal side of the scissile bond and exhibit sufficient specificity in their recognition sequence to avoid internal cleavage of the target protein. Factor Xa (Nagai et al., 1985Go) and enterokinase (LaVallie et al., 1993Go) are two of the most widely used cleavage systems on a laboratory scale, with expression vectors and enzymes commercially available from a variety of suppliers. Over the years, several improvements have been made to both systems. The proteases have been expressed as recombinant proteins and have themselves been fused to affinity tags to facilitate their removal after the cleavage reaction. An alternative system based on self-splicing intein sequences that does not require addition of a protease (Chong et al., 1997Go) is also commercially available. Regardless of the system used, successful cleavage defined by precision, specificity and efficiency depends on the overall conformation of the fusion protein, containing the three components: N-terminal fusion partner, cleavable linker and C-terminal fusion partner.

Here, we describe a novel cleavage method based on the maturation of the aspartic protease chymosin. Chymosin is the primary enzyme responsible for digestion of milk proteins in infant mammals and is widely used in the cheese industry. Like other aspartic proteases, chymosin has a bilobal structure with a substrate binding cleft to which each lobe contributes one catalytic aspartate residue (Tang, 1977Go). In addition to the cleavage of {kappa}-casein used in milk clotting, chymosin catalyzes its own precursor maturation. The maturation of the inactive precursor involves an activation step, elicited by low pH, followed by a processing step in which the N-terminal pro-peptide is cleaved (Figure 1A). Processing, which results in the formation of the mature protease (Foltmann, 1988Go), can occur through both intramolecular and intermolecular cleavage of the pro-peptide. The ‘heterologous cleavage’ method described here utilizes the chymosin pro-peptide (chypro) as a cleavable linker (Figure 1B). The precision of cleavage obtained with this method has been determined for four model proteins expressed in Escherichia coli. Furthermore, it was found that the precision of cleavage could be optimized by mutagenesis of the chymosin pro-peptide. The heterologous cleavage system was further tested with fusion proteins immobilized on an oilbody particle. Cleavage of immobilized fusion proteins has the advantage of combining the cleavage reaction and the removal of the undesired fusion partner into one step. It is a more rigorous test of the robustness of a cleavage system, because of potential limitations of accessibility.



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Fig. 1. Schematic representation of in vivo chymosin maturation (A) and ‘heterologous cleavage’ mediated by the chymosin pro-peptide (B). In vivo the zymogen pro-peptide is cleaved off when the molecule enters the low-pH environment of the stomach. Both intramolecular and intermolecular cleavage takes place. The heterologous cleavage is mediated by the intermolecular reaction.

 
Oleosins are structural proteins that are tightly associated with oilbodies, the natural oil storage organelles of plant seeds (Huang, 1996Go). They can be used as carriers and purification vehicles for industrial scale production of recombinant proteins (van Rooijen and Moloney, 1995Go; Moloney, 1997Go). The N- and C-termini of the oleosins are exposed on the surface of the oilbody, and the highly conserved hydrophobic central domain is embedded into the oilbody matrix (Parmenter et al., 1995Go; Huang, 1996Go). Proteins expressed in the seeds of transgenic plants as translational fusions with oleosins are correctly targeted to and tightly associated with the oilbody. Oilbodies and proteins associated with them can be easily separated from the majority of other seed cell components by flotation centrifugation (van Rooijen and Moloney, 1995Go; Moloney, 1997Go). Two of the model proteins, cystatin and thioredoxin, were expressed in Arabidopsis as oleosin fusions with chymosin pro-peptide between the fusion partners. These were used to test whether after isolation of the oilbodies, incubation with chymosin and flotation centrifugation, the mature proteins could be recovered in a purified form from the oilbody particle.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial expression

In-frame fusions of the 126 bp DNA fragment coding for chymosin pro-sequence (Foltmann et al., 1977Go) with hirudin variant 1 (Bagdy et al., 1976Go; Dodt et al., 1986Go), rice cystatin (Chen et al., 1992Go), Arabidopsis thioredoxin (Rivera-Madrid et al., 1993Go) and carp growth hormone (cGH) (Chao et al., 1989Go; Koren et al., 1989Go) were polymerase chain reaction (PCR)-amplified by overlap extension (Horton et al., 1993Go) and inserted into the NotI site of pGEX-4T-2 expression vector. The final constructs were verified by sequencing. Supplementary data are available at Protein Engineering online.

Bacterial expression vectors were transformed into E.coli cells BL21 (DE3). Cells were grown and induced with isopropyl-ß-D-thiogalactoside and the resultant glutathione S-transferase (GST) fusion proteins were purified according to the manufacturer’s protocol (Amersham Pharmacia Biotech).

Renaturation of GST-carp growth hormone from inclusion bodies

After cell lysis, inclusion bodies were collected by centrifugation, resuspended into 30 ml of distilled water and kept on ice, then 0.1 M sodium hydroxide solution was added slowly with constant stirring until the solution became transparent (~2 ml). A 4 ml volume of 10x phosphate-buffered saline was added and the solution was held on ice for 2–4 h. Subsequently, the solution was titrated to pH 8 by dropwise addition of 0.1 M HCl. The solution was centrifuged to remove particulate matter and the supernatant was applied to a glutathione–Sepharose column (Amersham Pharmacia Biotech).

Expression of fusion proteins in plant seeds

The plant transformation vector, pSBS was derived from the Agrobacterium binary plasmid pPZP (Hajdukiewicz et al., 1994Go). The gentamycin resistance gene of pPZP221 was replaced with a parsley ubiquitin promoter driving the transcription of phosphinothricine acetyltransferase gene and terminated by a parsley ubiquitin terminator sequence. This conferred resistance in transgenic plants to the herbicide glufosinate ammonium (Wohlleben et al., 1988Go). A promoter from the bean storage protein gene Phaseolin (Slightom et al., 1983Go) was used to drive the expression of the oleosin fusion proteins in a seed-specific manner. An origin of replication from pBR322 controls the replication of the plasmid in E.coli and Agrobacterium. A spectinomycin resistance gene confers antibiotic resistance to the bacterial hosts. In planta transformation of Arabidopsis was performed essentially as described by Clough and Bent (Clough and Bent, 1998Go). Oilbodies were isolated and washed by flotation centrifugation as described previously (van Rooijen and Moloney, 1995Go). Briefly, seed was ground with a mortar and pestle in 5 vol. of oilbody extraction buffer (0.5 M NaCl, 0.4 M sucrose, 50 mM Tris–Cl, pH 8.0). After centrifugation, the oilbodies were resuspended in high-stringency washing buffer (8 M urea, 100 mM Na2CO3) for the cystatin seed lines and in 50 mM Tris–Cl, pH 8.0, for the thioredoxin seed lines. The oilbodies were further washed by two subsequent centrifugation and resuspension steps in 50 mM Tris–Cl, pH 8.0.

Chymosin cleavage and analysis by SDS–PAGE

Cleavage reactions were performed in 100 mM sodium phosphate, pH 4.5, at 37°C for 2 h or at room temperature for 16 h. The optimal target-to-protease mass ratio was found to be 20:1 for GST-cGH fusions and 100:1 for all other fusions. Chymosin was provided by SKW Biosystems as a single-strength solution with 4 mg/ml protein and 83 milk clotting units/ml.

SDS–PAGE was run using standard protocols and stained with Coomassie Brilliant Blue R-250 (EM Science) (Sambrook and Russell, 2001Go).

Enzyme assays

Hirudin activity was assayed by the reduction of the rate of conversion of the chromogenic substrate N-p-tosyl-Gly-Pro-Arg p-nitroanilide (Sigma) by thrombin (Abildgaard et al., 1977Go; Lottenberg et al., 1981Go).

Cystatin activity was assayed by the inhibition of papain digestion of azocasein. The release of trichloroacetic acid-soluble, digested azocasein was measured spectrophotometrically (Zucker et al., 1985Go).

Preparation of samples for mass spectrometry

After completion of the cleavage reaction, the samples were incubated at 65°C (85°C for hirudin-containing fusions) for 5–10 min and centrifuged at 16 000 g to remove denatured chymosin, GST and uncleaved fusion protein. The supernatant was dialyzed against water in a Slide-a-lyzer cassette (Pierce) with a 3500 Da molecular weight cutoff membrane for 16 h at 4°C and dried down by freeze-drying.

For the recovery of cleavage products from oilbodies, the samples were first centrifuged at 16 000 g for 15 min to remove oilbodies and then the undernatant was subjected to heat denaturation, dialysis and freeze-drying as described above.

Mass spectrometry and N-terminal sequencing

Mass spectrometry was performed at Spectroscopy Laboratories, Plant Biotechnology Institute, NRC in Saskatoon on an AB Voyager MALDI/TOF mass spectrometer. N-terminal microsequencing was performed using Edman degradation of PVDF-immobilized proteins and was carried out by the Protein Chemistry Centre at the University of Victoria and the Biotechnology Laboratory at the University of British Columbia.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chymosin-mediated cleavage of hirudin-containing fusion proteins

Hirudin from medicinal leeches is a potent thrombin inhibitor with excellent pharmacological properties as a blood anticoagulant (Cheng-Lai, 1999Go). The interaction of the N-terminus of hirudin with the active cleft of thrombin is essential for inhibition. Substitutions or additions to the N-terminus of hirudin significantly reduce its inhibitory activity (Betz et al., 1992Go). We have used this characteristic as an indicator of cleavage accuracy and optimal cleavage conditions of a GST-chypro-hirudin fusion protein. As shown in Figure 2A, no inhibition of thrombin activity occurs when GST-chypro-hirudin or chymosin alone is added to a thrombin-catalyzed reaction. Thrombin activity is greatly reduced when GST-chypro-hirudin has been pre-incubated with chymosin, indicating the release of biologically active hirudin. This cleavage is specific for the chymosin pro-sequence, since a fusion protein with a Factor Xa cleavage site between GST and hirudin does not produce any hirudin activity (Figure 2A). The time course and pH dependence of the cleavage reaction are shown in Figure 2B and C, respectively. To purify the cleaved hirudin for further characterization, we took advantage of its heat stability. Whereas chymosin, GST and GST-chypro-hirudin become denatured and precipitate when incubated at 85°C, hirudin remains soluble. Cleaved hirudin purified in this fashion was subjected to analysis by mass spectrometry. As shown in Figure 3A (top profile), two major molecular ions are apparent in the mass profile. The 6968 Da peak corresponds to the mass of native hirudin, and the larger 7260 Da peak corresponds to hirudin with three extra amino acids on the N-terminus, which by deduction originate from the pro-sequence (Figure 4).



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Fig. 2. Anti-thrombin activity of cleaved hirudin fusion proteins. The conversion of the chromogenic substrate N-p-tosyl-Gly-Pro-Arg p-nitroanilide by thrombin is measured spectrophotometrically over a period of 3 min. Hirudin activity is measured as the decrease in the rate of substrate conversion: {Delta}A405/min = A405/min (blank) – A405/min (sample) and converted into hirudin concentration using a standard curve. All measurements are an average of at least three replicate experiments. (A) Release of hirudin activity by chymosin cleavage of GST-chypro-hirudin. (B) Time course of the chymosin cleavage reaction. GST-chypro-hirudin was incubated with chymosin at 37°C and aliquots were assayed for hirudin activity at time intervals. (C) pH dependence of the chymosin cleavage reaction. GST-chypro-hirudin was incubated with chymosin in 100 mM sodium phosphate of varying pH. After 2 h at 37°C, the samples were assayed for hirudin activity.

 


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Fig. 3. Mass spectrometric profiles (m/z) of purified recombinant protein after cleavage of the fusion proteins with chymosin. (A) Cleavage of bacterially produced GST-chypro-hirudin and pro-peptide mutants. Full-length hirudin shows a peak at 6968 Da and at half mass 3485 Da as a result of double ionization. The larger peaks at 7260 for wild-type chypro and 7819 and 8606 for Y39S correspond to cleavage sites within the chymosin pro-peptide. The peak at 5836 corresponds to a nine amino acid C-terminal truncation of hirudin as a result of over-digestion by chymosin. (B) Cleavage of oilbody-associated oleosin-{Delta}YSG chypro-cystatin. The deduced molecular weight for cystatin is 11 391 Da. The minor peak with a mass of 12 038 corresponds to cleavage at position –9 in the chypro sequence and is a result of incomplete cleavage.

 


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Fig. 4. Protein sequence of the GST-chypro-hirudin junction and chypro mutants. The 42 amino acids that comprise the chymosin pro-peptide are underlined. Arrows indicate the cleavage sites as determined by mass spectrometry.

 
Mutagenesis of the chymosin pro-peptide

To determine whether cleavage at the upstream site could be eliminated, we used both a deletion and a substitution approach. Expression vectors were constructed in which the nucleotides coding for the three amino acids interposed between the two cleavage sites were deleted, giving rise to two new pro-peptide configurations called {Delta}YSG and {Delta}SGF. In the third modification, Y39S, the tyrosine codon was substituted by a serine codon. Cleavage of the expressed mutant fusion proteins by chymosin resulted in the release of active hirudin. Mass analysis of the cleaved products determined by mass spectrometry, showed that only precise cleavage at position +1 occurs for the deletion mutants {Delta}YSG and {Delta}SGF, while alternative cleavage sites at position –9 and –15 appeared in the Y39S mutant (Figures 3A and 4).

Heterologous cleavage of other fusion partners

To demonstrate that the heterologous cleavage system can be applied to a wider variety of proteins, rice cystatin, Arabidopsis thioredoxin and cGH were expressed as GST-chypro- and GST-{Delta}YSG chypro- fusions in E.coli. Like hirudin, cystatin and thioredoxin fusions were expressed in soluble form and could be purified directly from cell lysates. Carp growth hormone was found predominantly in inclusion bodies and solubilization at alkaline pH followed by neutralization was required prior to affinity purification. SDS–PAGE analysis of cleavage products of GST-chypro-thioredoxin and GST-chypro-cGH is shown in Figure 5A and B, respectively. The cleaved products were further characterized by mass spectrometry for cystatin and thioredoxin and by N-terminal sequencing for cGH. The results, summarized in Table I, show that cleavage of cystatin and thioredoxin occurs at sites identical with those found for hirudin: at the junction between the pro-peptide and the mature protein and three amino acids upstream with the wild-type pro-peptide and predominantly at the pro-peptide–mature protein junction with the {Delta}YSG mutated sequence.



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Fig. 5. SDS–PAGE analysis of fusion protein cleavage products. Coomassie Blue-stained gels show cleavage of GST-chypro-thioredoxin (A) and GST-chypro-cGH (B). (A) 5 µg uncleaved GST-chypro-thioredoxin (lane 1) and GST-{Delta}YSG chypro-thioredoxin (lane 3) fusion protein; 20 µg GST-chypro-thioredoxin (lane 2) and GST-{Delta}YSG chypro-thioredoxin (lane 4) cleaved with chymosin; cleaved, heat purified thioredoxin from GST-{Delta}YSG chypro-thioredoxin (lane 5). (B) Uncleaved GST-chypro-cGH, GST-{Delta}YSG chypro-met-cGH and GST-{Delta}YSG chypro-cGH (lanes 1, 2, 3); cleaved GST-chypro-cGH, GST-{Delta}YSG chypro-met-cGH and GST-{Delta}YSG chypro-cGH (lanes 5, 6, 7); mature cGH produced in E.coli inclusion bodies (lane 4). In both panels top arrows (F) indicate the intact fusion protein. Middle (N) and bottom arrows (C) indicate the N-terminal (GST) and C-terminal fusion partners, respectively. Location of molecular weight standards is indicated in kDa.

 

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Table I. Summary of chymosin cleavage sites of wild-type- and pro-sequence mutants in combination with various fusion partners as determined by mass spectrometry and N-terminal sequencing
 
Cleavage of cGH occurred exclusively at the upstream –3 position when it was fused to wild-type chypro peptide and an alternate cleavage site further upstream (position –9) was found with the {Delta}YSG pro-peptide. Mature cGH protein starts with the amino acid serine. We speculated that this proximal serine prevents correct cleavage. To test this hypothesis, a mutant was generated in which a methionine codon was inserted at the junction of {Delta}YSG pro-peptide and cGH sequence. In the resulting fusion protein, methionine is the first amino acid downstream of the desired cleavage site. The cleavage, as determined by N-terminal sequencing, occurred at the predicted +1 position, releasing cGH with a methionine at its N-terminus (Figure 5B and Table I).

Cleavage of oilbody-associated fusion proteins

The utility of the heterologous cleavage system was further analyzed with oilbody-associated oleosin fusions to assess the applicability of this system to immobilized proteins. Expression vectors that drive the seed-specific expression of oleosin-chypro-cystatin, oleosin-{Delta}YSG chypro-cystatin and oleosin-{Delta}YSG chypro-thioredoxin were introduced into Arabidopsis. Seed from transgenic plants expressed and correctly targeted the fusion proteins to the oilbody (Figure 6A for cystatin; data not shown for thioredoxin). After isolation, the recombinant oilbodies were treated with chymosin under the same conditions as determined for the bacterial GST fusion proteins. The efficiency of cleavage was determined by densitometry of fusion and cleaved protein in SDS–polyacrylamide gels. The cleavage efficiency of the oilbody-associated fusion protein was found to be comparable to the efficiency of cleavage of the soluble bacterial fusions and was >90% (Figure 6A). After removal of the oilbodies by centrifugation, the undernatant contained predominantly the cleaved product (Figure 6A, lane 5). Based on protein content and densitometry of Coomassie Blue-stained gels, an estimated 98% of native seed protein has been removed at this point. N-terminal sequencing of cleaved cystatin and thioredoxin showed that the cleavage pattern was identical with that obtained for bacterial fusions, i.e. cleavage at the correct site and three amino acids upstream with the wild-type pro-peptide and exclusively at the correct site with {Delta}YSG chypro, respectively (Table I). Mass spectrometry confirmed the correct size of mature cystatin, ruling out any heterogeneity of the C-terminus (Figure 3B). The purified cystatin was biologically active as an inhibitor of the protease papain (Figure 6B).



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Fig. 6. Chymosin cleavage of oilbody-associated oleosin-{Delta}YSG chypro-cystatin. (A) SDS–PAGE analysis visualized by Coomassie Blue staining; 20 µg oilbody protein/lane isolated from Arabidopsis seed of a wild-type (lane 2) and transgenic line that expresses oleosin-{Delta}YSG chypro-cystatin (lane 3). The four oleosin isoforms give rise to the predominant 18 kDa band and the less abundant 21 and 23 kDa bands; 20 µg cleaved oilbodies (20 µl cleavage reaction containing 1 µg/µl oilbody protein) expressing oleosin-{Delta}YSG chypro-cystatin (lane 4); undernatant of the chymosin cleavage reaction. The reaction from lane 4 was centrifuged to remove oilbodies. The undernatant contains predominantly full-length, mature cystatin (lane 5). Lane 1, low molecular weight standards (Amersham Pharmacia Biotech). F, intact fusion protein; N, N-terminal fusion partner, oleosin; C, C-terminal fusion partner, cystatin. (B) Inhibition of papain activity by cystatin cleaved from oilbody-associated oleosin-{Delta}YSG chypro-cystatin. Papain digestion of azocasein into trichloroacetic acid-soluble fraction is measured by absorbance at 450 nm.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cleavable linkers are generally derived from a substrate sequence that is susceptible to proteolytic cleavage (Walsh and Swaisgood, 1996Go). The heterologous cleavage system described here utilizes a pro-peptide of an autocatalytically maturing protease. The chymosin pro-peptide is sufficient to direct cleavage predominantly to the junction of the pro-peptide and the heterologous protein.

The time-scale of the cleavage reaction (Figure 2B) is comparable to that of enterokinase and Factor Xa cleavage systems. The pH optimum (Figure 2C), on the other hand, is more acidic than that for enterokinase and Factor Xa. It is well suited for the cleavage of acid-stable proteins such as the cytokines transforming growth factor-{alpha}. (Todaro et al., 1980Go) and type I interferons (Martal et al., 1998Go). The optimum pH for autocatalytic maturation of chymosin is 2. This coincides with the physiological environment where chymosin is active – the calf stomach. The optimum for heterologous cleavage, defined as the conditions resulting in maximum recovery of authentic product, determined here, is pH 4.5 (Figure 2C). The thrombin inhibition assay employed in determining the optimum conditions relies both on precise cleavage and the presence of full-length hirudin molecules. Cleavage carried out at lower pH resulted in very efficient, but non-specific, digestion of the fusion protein as visualized by the disappearance of the fusion protein that is not accompanied by the appearance of full-length protein on SDS–PAGE gels (data not shown). It is probable that at low pH the fusion protein partially unfolds and becomes more susceptible to non-specific cleavage. By increasing the pH, chymosin proteolytic activity becomes restricted to the most sensitive site, which is the junction between the pro-sequence and hirudin (Hubbard, 1998Go). Chymosin remains active up to pH 6.

Authenticity of primary protein sequence is an important factor in the production of pharmaceutical proteins. Using the wild-type pro-peptide, two cleavage sites are apparent: the desired site at the pro-peptide–recombinant protein junction and an alternative site within the pro-peptide. Based on the similarity of prochymosin to the crystal structure of propepsin (Bateman et al., 1998Go), the comparison of pro-peptides of several related aspartic proteases (Foltmann, 1988Go; Koelsch et al., 1994Go) and on the studies of the crystal structure of free and bound chymosin binding pocket (Groves et al., 1998Go), mutagenesis of the prosequence was performed. This resulted in the elimination of the alternative cleavage site for the {Delta}YSG and {Delta}SGF mutants. There is some restriction as to the N-terminal amino acid sequence of the recombinant protein that will allow precise cleavage. Hirudin, cystatin and thioredoxin, with first amino acids valine, methionine and methionine, respectively, were cleaved precisely (Table I), whereas mature cGH with an N-terminal serine, did not show precise cleavage. Insertion of a methionine as the N-terminal residue restored precise cleavage, suggesting that it is the first residue that is important for precise cleavage and that some amino acids negatively affect the precision of the cleavage reaction. Modified specificity due to downstream sequences has also been shown for Factor Xa (Nagai and Thogersen, 1987Go; He et al., 1993Go) and enterokinase (Collins-Racie et al., 1995Go). In order to restore the authentic protein sequence for cGH using the heterologous cleavage system, the N-terminal methionine could, if needed, be removed post-cleavage by digestion with methionyl aminopeptidase (Hirel et al., 1989Go; Dalboge et al., 1990Go).

The heterologous cleavage system was employed for the cleavage of oilbody-associated fusion proteins produced in Arabidopsis. The efficiency and precision of cleavage were found to be identical with those for the cleavage of soluble fusion proteins expressed in bacteria, suggesting that the proximity of the oilbody does not pose any steric constraints on the interaction of the protease and its substrate. On the other hand, based on the activity of cleaved hirudin, cleavage of oleosin-hirudin separated by a Factor Xa cleavage site from transgenic oilbodies (Parmenter et al., 1995Go) was 50 times less efficient compared with the cleavage of GST-Factor Xa-hirudin expressed in bacteria (data not shown). The mutated chymosin pro-peptide is 39 amino acids in length, compared with four amino acids that constitute the Factor Xa cleavage site. It may be that the length of the chymosin pro-peptide positions the cleavage site further away from the oilbody surface, thus providing better access for the cleavage agent.

This work demonstrates that chymosin can be used as an efficient cleavage enzyme for a variety of heterologous fusion proteins including those immobilized on particles such as oilbodies. The efficiency and precision of this system appear to be superior to those of many of the routine cleavage methods used on a laboratory scale. Although the chymosin used in these studies was of bovine origin, we have successfully repeated the experiments with plant-produced bovine chymosin (van Rooijen et al., 2001Go). This approach will permit the use of fusion proteins for larger scale manufacturing, both by reducing the cost of the cleavage agent and by addressing safety issues arising from the use of cleavage agents of mammalian origin.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received June 5, 2003; revised August 1, 2003; accepted August 20, 2003.





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