Extracellular expression and affinity purification of L-asparaginase from E. chrysanthemi in E. coli
© Karamitros and Labrou 2014
Received: 25 April 2014
Accepted: 24 July 2014
Published: 20 August 2014
L-asparaginases (E.C.22.214.171.124, L-ASNases) are successfully used as anti-neoplastic agents in the chemotherapy of acute lymphoblastic leukemia (ALL) and therefore are of high interest for the medical and scientific community. In the present study we report the recombinant, extracellular expression and affinity purification of L-asparaginase from Erwinia chrysanthemi 3937 (ErL-ASNase) in E. coli.
Recombinant E. coli strains were screened for their ability to express and translocate ErL-ASNase to the culture medium. The strain E. coli Rosetta (DE3) exhibited the highest extracellular expression levels among all the strains tested and it was chosen for further optimization and the development of purification protocol. Affinity adsorbents with immobilized L-Asn, L-Asp and L-Glu were synthesized by solid-phase chemistry and evaluated for their ability to bind and purify ErL-ASNase directly from the culture medium. The affinity adsorbent with immobilized L-Asp (L-Asp-Sepharose CL-6B) showed the highest purifying ability for ErL-ASNase. Adsorption equilibrium studies revealed that the adsorption of ErL-ASNase follows Langmuir isotherm with KD = 0.21 μM and maximum binding capacity 4.7 mg enzyme/g moist wet adsorbent. This affinity adsorbent was used for the development of one-step purification protocol. The proposed protocol is simple, fast, gentle and afforded ErL-ASNase with high purity and yield.
We show that the recombinant expression of ErL-ASNase in E. coli results in the secretion to the culture medium due to the presence of its natural peptide leader at the N-terminus. We developed an L-Asp-based affinity adsorbent which allowed the purification of the enzyme in one step, achieving high purity levels. This approach is advantageous over the other conventional tag-based purification methods, which require additional treatment steps for the cleavage and isolation of the affinity tags. Overall, the strategy employed for expression and purification of this protein drug uses green chemistry principles allowing the reduction of processing time and purification steps, making the approach more sustainable and attractive.
KeywordsL-asparaginase Enzyme purification Extracellular expression Leukemia Therapeutic enzymes
L-asparaginase (E.C.126.96.36.199, L-ASNase) catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia. The enzyme attracts attention due to its efficient use in the therapy of Acute Lymphoblastic Leukemia (ALL) –. L-ASNase is also used in combination with other drugs for the treatment of other type of malignancies such as non-Hogkins Lymhoma, chronic lymphoblastic leukemia, lymphosarcoma, melanosarcoma ,. In the case of ALL the therapeutic function of L-ASNase is due to the fact that the asparagine synthetase of the leukemic lymphoblasts is downregulated resulting in deficiency of L-asparagine synthesis –. Therefore, the survival of those cells is dependent on the exogenous supply of L-asparagine from the blood. L-ASNase depletes the available L-Asn in the blood, causing inhibition of protein synthesis and apoptosis to the malignant cells ,. It must be underlined that the normal cells are not affected since they can synthesize L-Asn.
L-ASNase is the only bacterial enzyme that has been approved by the FDA for human therapy . The two approved preparations are from E. coli and E. chrysanthemi. The E. coli ASNase is the first choice drug as it is capable of leading to remission very fast and causing relatively mild toxic effects . Nevertheless, in cases where immunogenicity and allergic side effects arise due to E. coli L-ASNase, treatment is immediately switched to the Erwinia enzyme ,. It has been reported, that the two enzymes exhibit totally different immunogenicity patterns in patients .
Affinity chromatography is considered to be one of the most refined and efficient techniques, which has been widely used in downstream processing with outstanding success in certain cases . In this technique, the molecule of interest binds to another immobilized molecule, most often called the ligand. Ideally, the ligand has such structural and physicochemical properties that it is recognized specifically only by the target biomolecule . As a result, the final purity depends on the specificity between the ligand and the biomolecule as well as, the strength of their interaction .
Periplasmic protein expression and potentially, extracellular secretion in the growth medium facilitates enormously the downstream processing protocol due to lower level of extracellular expressed proteins compared to intracellular ones –. As a result, higher protein purity can be achieved applying less number of chromatographic steps.
The long-term usage of L-asparaginase leads to life-threatening multiple toxic effects. Among its multiple toxic effects, L-asparaginase induces allergic reactions usually due to low purity enzyme preparations and the presence of unwanted contaminations by bacterial proteins. Therefore, efficient recombinant expression, coupled with simple, rapid and low cost purification protocol would assist the overall production scheme of such an important enzyme, whose purity is extremely critical due to its use for human treatment. The aim of the present work is to study the optimization of the extracellular expression of ErL-ASNase in E. coli by attaching its natural peptide leader at the N-terminus. The secreted recombinant L-ASNase was ultimately purified by applying an efficient one-step affinity chromatographic protocol.
L-Asn, L-Asp, L-Glu, trichloroacetic acid, 1,4-butanediol diglycidyl ether and crystalline bovine serum albumin (fraction V) were obtained from Sigma-Aldrich (U.S.A.). Sepharose CL-6B were purchased from Pharmacia. Nessler’s reagent was obtained from Fluka (Germany). Isopropyl-beta-D-thiogalactoside (IPTG) was from Genaxis (U.K.). Yeast extract, peptone, agar and glycerol were purchased from Scharlau (Spain).
Solid phase synthesis of the affinity adsorbents
Synthesis of the affinity adsorbents was carried using a two-step procedure as follows: i) Activation of agarose: Sepharose CL-6B beads (5 g) were thoroughly washed with double distilled water in a glass filter funnel and drained. The washed beads were suspended in 1M NaOH solution (5 mL) and shaken on a rotary shaker (140 rpm, 25°C, 120 min). The beads were washed with double distilled water to remove excess NaOH and 1,4-butanediol diglycidyl ether (5 mL) was added to the Sepharose CL-6B. The mixture was shaken at 140 rpm, 25°C for 12 h. After completion of the reaction the 1,4-butanediol diglycidyl ether-activated Sepharose CL-6B was washed with double distilled water.
ii) Coupling with the ligand (L-Asp, L-Glu, or L-Asn): Activated Sepharose CL-6B (5 g) was resuspended in 20 mL KH2PO4, pH 7.5, along with either L-Asp or L-Glu or L-Asn (1 g dissolved in 10 mL Na2CO3, pH 10). The mixture was shaken on a rotary shaker (210 rpm, 25°C) for 64 h. After completion of the reaction, the gel was filtered, washed with distilled water and stored in water at 4°C.
Optimization of extracellular expression of ErL-ASNase in E. coli
E. coli strains used in the present study
endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44 F'[ ::Tn10 proAB+ lacIq Δ(lacZ)M15] hsdR17(rK− mK+)
F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ−
F− ara-14 leuB6 secA6 lacY1 proC14 tsx-67 Δ(ompT-fepC)266 entA403 trpE38 rfbD1 rpsL109 xyl-5 mtl-1 thi-1
New England Biolabs
F− ompT gal dcm lon hsdSB(rB− mB−) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])
F− ompT gal dcm lon hsdSB(rB− mB−) λ(DE3),(cmR)
F− ompT hsdSB(rB− mB−) gal dcm (DE3) pRARE (CamR)
F− ompT gal dcm lon hsdSB(rB− mB−) λ(DE3) pLysS(cmR)
Enzymatic activity assay
Enzyme assays were performed at 37°C by measuring the amount of ammonia liberated upon reaction with Nessler’s reagent. Activities were measured as described by Kotzia & Labrou . One unit of L-ASNase activity is defined as the amount of enzyme that liberates 1 μmol of ammonia from L-Asn per min at 37°C. Protein concentrations were determined at 25°C by the method of Bradford  using bovine serum albumin (fraction V) as standard.
Screening of affinity adsorbents for ErL-ASNase binding
Columns packed with affinity adsorbents (2 mL) were washed with 50 mL double distilled H2O and equilibrated with 30 mL of 20 mM KH2PO4, pH 7.5. Culture medium supernatant containing the secreted ErL-ASNase (approximately 80 U) was dialysed overnight against 20 mM KH2PO4 pH 7.5 and was loaded onto each column. Non-adsorbent proteins were washed off with 30 mL of equilibration buffer. The adsorbent’s binding capacity was determined as the proportion (%) of L-ASNase (units) bound on the affinity adsorbent to the overall units loaded.
Adsorption equilibrium studies
In a total volume of 1 mL of 20 mM KH2PO4, pH 7.5, varying amounts of purified L-ASNase, previously dialysed in 20 mM KH2PO4 pH 7.5, were mixed with 3 mg of affinity adsorbent (L-Asp-Sepharose CL-6B). The suspensions were shaken for 120 min in order for the system to reach equilibrium. The suspension was then centrifuged (4000g, 2 min) and the amount of unbound protein in the supernatant was determined by the Bradford method . Bound protein was calculated by subtracting the amount of unbound protein from the total amount of protein added. The data were analyzed using Igor Pro software platform (Wavemetrics Co.).
Purification of extracellular ErL-ASNase on immobilized L-Asp affinity adsorbent
Culture medium (10 mL) was dialyzed overnight at 4°C against binding buffer (20 mM KH2PO4, pH 7.5) containing different concentrations of NaCl (0.05-0.2 M) and adjusted to different pH values (7.5-8.6) and loaded onto L-Asp adsorbent, previously equilibrated with 30 mL of the binding buffer. The column was kept closed for 10 min for the system to reach equilibrium. Subsequently, the column was exhaustively washed either with binding buffer containing 2 M NaCl or with ice-cold ddH2O. Finally, bound ErL-ASNase was eluted with 10 mL of 20 mM L-Asp dissolved in 20 mM potassium phosphate buffer, pH 7.5. Both flow-through and washing fractions were kept and the protein content was determined by Bradford and analyzed by SDS-PAGE and activity assays.
SDS polyacrylamide gel electrophoresis was performed according to the method of Laemmli  on a slab gel containing 12.5% (w/v) polyacrylamide (running gel) and 2.5% (w/v) stacking gel. The protein bands were stained with Coomassie Brilliant Blue R-250.
Results & discussion
Optimization of extracellular expression of ErL-ASNase in E. coli
Synthesis and screening of affinity adsorbents for ErL-ASNase binding
Binding (%) of ErL-ASNase to the affinity adsorbents
L-Asn-Sepharose CL 6B
L-Asp-Sepharose CL 6B
L-Glu-Sepharose CL 6B
Adsorption equilibrium studies
Purification of ErL-ASNase on immobilized L-Asp affinity adsorbent
Given the existing high interest in L-ASNase serving as a neoplastic agent and being a milestone for the treatment of ALL, we focused on the development of an efficient protocol for the expression and purification of this protein drug. The achievement of high purity levels in the case of pharmaceutical proteins is of great importance and plays a vital role in therapeutic applications ,. Therefore, it is a challenging task to establish an efficient protocol not only for the recombinant expression of a therapeutic protein, but also for its purification, which must be simple, fast, mild and of low cost. Recently, L-ASNase from E. coli was extracellular expressed by fusing the gene coding for 6-His-tagged L-ASNase to the pelB leader sequence. In that work the recombinant 6-His-tagged protein was purified from the culture supernatant in a single step using Ni-NTA affinity chromatography ,. However, this approach necessitates additional purification steps such as the cleavage and the removal of the 6-His-tag, which add to the overall cost of the purification process.
In the present work we showed that recombinant ErL-ASNase is efficiently expressed in E. coli Rosetta(DE3) and secreted into the culture medium. The expression levels were optimized by testing different E. coli strains, culture media, as well as temperature and incubation times. The secreted recombinant protein was purified directly from the culture medium, thereby simplifying considerably the downstream processing. The purification protocol was based on affinity adsorbent with immobilized L-Asp as a ligand, which exhibited high specificity and affinity for ErL-ASNase. The final purity of the enzyme (as judged by SDS-PAGE) justifies the potential for scaling up the whole process, taking also into account the low-cost materials used.
Acute lymphoblastic leukaemia
L-asparaginase from Erwinia chrysanthemi 3937
Food and drug administration
Polyacryamide gel electrophoresis
Sodium dodecyl sulfate
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