Expression optimization of recombinant cholesterol oxidase in Escherichia coli and its purification and characterization
AMB Express volume 8, Article number: 183 (2018)
Cholesterol oxidase is a bacterial flavoenzyme which catalyzes oxidation and isomerization of cholesterol. This enzyme has a great commercial value because of its wide applications in cholesterol analysis of clinical samples, synthesis of steroid-derived drugs, food industries, and potentially insecticidal activity. Accordingly, development of an efficient protocol for overexpression of cholesterol oxidase can be very valuable and beneficial. In this study, expression optimization of cholesterol oxidase from Streptomyces sp. SA-COO was investigated in Escherichia coli host strains. Various parameters that may influence the yield of a recombinant enzyme were evaluated individually. The optimal host strain, culture media, induction time, Isopropyl ß-d-1-thiogalactopyranoside concentration, as well as post-induction incubation time and temperature were determined in a shaking flask mode. Applying the optimized protocol, the production of recombinant cholesterol oxidase was significantly enhanced from 3.2 to 158 U/L. Under the optimized condition, the enzyme was produced on a large-scale, and highly expressed cholesterol oxidase was purified from cell lysate by column nickel affinity chromatography. Km and Vmax values of the purified enzyme for cholesterol were estimated using Lineweaver–Burk plot. Further, the optimum pH and optimum temperature for the enzyme activity were also determined. We report a straightforward and easy protocol for cholesterol oxidase production which can be performed in any laboratory.
Cholesterol oxidases (EC 126.96.36.199) are bifunctional bacterial flavoenzymes belonging to the family of oxidoreductase which catalyze the first step in the catabolism of cholesterol. They catalyze oxidation as well as isomerization of cholesterol and produce equimolar amounts of cholest-4-en-3-one coupled with hydrogen peroxide as the final products (Moradpour and Ghasemian 2016). There are two types of cholesterol oxidase (ChO) depending on the nature of the bond between FAD cofactor and apoenzyme. In type I, the FAD cofactor is linked to the protein through a noncovalent bond, while in type II, the cofactor is covalently bond to the apoenzyme (Vrielink and Ghisla 2009). Both types of enzymes have found wide applications as a useful biotechnological tool.
Cholesterol oxidase is the second most widely used enzyme in clinical laboratories (Doukyu et al. 2009). This enzyme is commonly used for determining cholesterol levels both in serum and in other biological samples (MacLachlan et al. 2000). On the other hand, the ability of cholesterol oxidase in bioconversion of 3β-hydroxysteroids makes it a valuable enzyme for transformation of sterols and non-sterols in the pharmaceutical industry (Doukyu 2009). Recently, many attempts have been made to reduce cholesterol levels in foods. The reduction of food cholesterol levels may occur via enzymatic methods (Yehia et al. 2015). Many experiments have been conducted to reduce milk and yolk cholesterol levels using cholesterol oxidase (Lv et al. 2002; Serajzadeh and Alemzadeh 2010; Smith et al. 1991). In addition, other investigations have addressed the role of cholesterol oxidase as an approach to pest control strategies (Cho et al. 1995; Purcell et al. 1993).
ChO has no mammalian homolog and is totally produced by pathogenic and nonpathogenic bacteria. Pathogenic bacteria employ this enzyme for infection of host macrophages by oxidation of membrane cholesterol, while nonpathogenic bacteria tend to utilize ChO as a metabolic tool for obtaining carbon sources from cholesterol decomposition (Pollegioni et al. 2009). So far, many efforts have been made to obtain the ChO from original microorganisms. Nevertheless, this approach suffers from some challenges such as difficult growth conditions and low productivity of original microorganisms (MacLachlan et al. 2000). In order to find a solution for these issues, ChO genes from different bacterial sources have been cloned and expressed which would be effective for commercial application of enzyme production (Brigidi et al. 1993; Corbin et al. 1994; Fujishiro et al. 1990; Horii et al. 1990; Liu et al. 1988; Molnár et al. 1991; Murooka et al. 1986; Nishiya et al. 1997; Ohta et al. 1992; Purcell et al. 1993; Solaiman and Somkuti 1991, 1995; Solaiman et al. 1992; Somkuti et al. 1991, 1995; Somkuti and Solaiman 1997). ChO from Streptomyces sp. SA-COO (ChOA) secretory production has been proved in a Streptomyces host-vector system (Murooka et al. 1986). Also, the ChOA gene has been cloned and sequenced (Ishizaki et al. 1989). Nomura et al. successfully expressed the ChOA gene in Escherichia coli (Nomura et al. 1995). Further, the thermal stability of the ChOA was improved in another study (Nishiya et al. 1997).
Recombinant ChOA production in a large quantity facilitates its biochemical characterization and its use in industrial processes. To this end, in the current study, we have taken a straightforward and effective approach to maximize ChOA production by optimizing the culture and induction parameters in shaking flasks.
Materials and methods
Strains, materials, and culture media
Escherichia coli host strains BL21(DE3), BL21(DE3)pLysS, and Rosetta-gami2(DE3) were obtained from Novagen (Madison, WI, USA). Synthesis of plasmid pET24b-ChOA was ordered to Bio Basic Inc. (ON, Canada). Ni-CAM HC Resin, isopropyl-β-d-thiogalactopyranoside (IPTG), kanamycin and chloramphenicol were purchased from Sigma-Aldrich (MO, USA). All other chemicals were prepared from Merck chemical company (Darmstadt, Germany). The following liquid media were used: Luria–Bertani (LB, 10 g/L peptone, 5 g/L yeast extract, 5 g/L NaCl, Merck), Super Broth (SB, 32 g/L peptone, 20 g/L yeast extract and 5 g/L NaCl, Merck), Terrific Broth (TB, 12 g/L peptone, 24 g/L yeast extract, 8 g/L glycerol, 17 mM KH2PO4 and 72 mM K2HPO4, Merck).
Optimization of recombinant ChOA expression
Expression of ChOA in different E. coli hosts
Initially, three different E. coli strains capability for the production of recombinant ChOA were assessed under our routine laboratory conditions. At first, ChoA gene (GenBank accession number M31939) was designed into pET24b(+) expression plasmid between NdeI-BamHI restriction sites (GenBank accession number MH810339). Then, 1 µL of pET24-ChOA plasmid was transformed into chemically competent cells of BL21(DE3), BL21(DE3)pLysS, and Rosetta-gami2(DE3) host strains. We used 50 µg/mL kanamycin in the solid and liquid medium of each of the three strains and additional 25 µg/mL chloramphenicol in the case of BL21(DE3)pLysS and Rosetta-gami2(DE3). After overnight incubation, a single colony of each strain was taken from LB agar plates and used for inoculation of 3 mL pre-culture media and incubated at 37 °C, 160 rpm for 12 h. On the following day, 10 mL of LB media was inoculated by 100 µL of pre-culture media and incubated under the same conditions. When the optical density at 600 nm (OD600nm) reached 0.6, IPTG was added up to a final concentration of 0.5 mM. The cells were harvested after 6 h by centrifugation at 7000×g, 4 °C, and within 10 min. The harvested cells were resuspended in 0.5 mL of PBS buffer containing NaCl (0.3 M) at pH 7. Bacterial cells were disrupted by sonication and the lysate was centrifuged at 13,000×g, 4 °C, within 20 min. The productivity of each host strain was evaluated by enzyme activity assay in the crude extract. ChOA activity was measured at 25 °C by a modification of the method of Allain et al. (1974) and Doukyu et al. (2008). The assay mixture contained 100 mM potassium phosphate pH 7.0, 1 mM cholesterol, 21 mM phenol, 1.4 mM 4-aminoantipyrine and 5 U/mL peroxidase. The reaction was started by addition of 100 µL sample to 1 mL assay mixture and the appearance of the red chromophore was monitored continuously at 500 nm. Blanks without enzyme or without cholesterol were routinely run in parallel. One unit of activity was defined as the formation of 1 µmol of hydrogen peroxide (0.5 µmol of quinoneimine dye) per min at 25 °C.
Culture media optimization
To determine the optimal culture media, the overnight culture of BL21(DE3)pLysS harboring pET24-ChOA plasmid was made in 3 mL of LB media. Then, 10 mL of three different medium types including LB, TB, and SB were inoculated with a pre-culture with the ratio of 1:100. When OD600nm reached 0.6, the cultures were induced with 0.5 mM IPTG and incubated at 37 °C, 160 rpm for 6 h. The cultures were harvested and the pellet was resuspended in 0.5 mL of PBS buffer. After sonication, the cell lysate was centrifuged at 13,000×g, 4 °C, for 20 min. The total activity of recombinant ChOA was measured by performing enzyme assay in the supernatant crude extract to determine productivity.
Optimum induction time
BL21(DE3)pLysS cells containing pET24-ChOA were grown overnight in LB media. Fresh culture (4 flasks) containing 10 mL TB media was inoculated (1:100) and incubated at 37 °C, 160 rpm. When the OD600nm of cultures reached 0.3, 0.6, 1.2 and 1.8, induction was made with 0.5 mM IPTG. Each culture was incubated for 6 h at 37 °C, 160 rpm. The harvested cells were resuspended in 0.5 mL of buffer (PBS, pH 7) and disrupted by sonication, then centrifuged at 13,000×g, 4 °C, for 20 min. Quantification of active (soluble) enzyme was performed by enzyme activity assay.
Optimum IPTG concentration
The effects of various IPTG concentrations on ChOA productivity were further evaluated. For this purpose, five flasks containing 10 mL of TB media were inoculated by a pre-culture with the ratio of 1:100. The cultures were incubated at 37 °C, 160 rpm until OD600nm reached 0.6. The cell cultures were induced by IPTG concentrations of 0.05, 0.1, 0.25, 0.5, and 1 mM respectively. After disruption and centrifugation of harvested cells, enzyme expression was measured by enzyme activity assay.
Induction temperature and post-induction incubation time
The productivity of recombinant ChOA was evaluated at different incubation temperatures (15 °C, 25 °C, and 37 °C), as well as four different post-induction incubation times (6, 8, 16, and 24 h). These parameters were investigated in three flasks containing 20 mL of TB media, inoculated by 0.2 mL of pre-cultured BL21(DE3)pLysS harboring ChOA gene. The induction was done at OD600nm ≃ 0.6 by adding IPTG in a final concentration of 0.25 mM. After the induction, the flasks were incubated at 15 °C, 25 °C, and 37 °C on a rotary shaker with a speed of 160 rpm. In order to determine the optimal post-induction incubation time, 2 mL of culture media from each flask was withdrawn at different time (6, 8, 16, and 24 h) intervals. The collected samples were centrifuged and pellets were resuspended in the buffer, and then the cells were disrupted by sonication. Once the samples were prepared, enzyme activity assay performed for quantification of the expressed recombinant enzyme.
Large-scale expression of ChOA under optimized condition
Overexpression of ChOA gene was performed according to the results of optimized protocol. A pre-culture was made by inoculating 5 mL of LB media containing kanamycin (50 µg/mL) and chloramphenicol (25 µg/mL) with pET24-ChOA harboring BL21(DE3)pLysS cells. Then, 500 mL of TB media containing 50 µg/mL kanamycin and 25 µg/mL chloramphenicol was inoculated by the pre-culture. When OD600nm reached 0.6, induction of ChOA gene expression was done by adding IPTG up to a final concentration of 0.25 mM and continued with 24 h incubation at 15 °C, 160 rpm. The harvested bacterial pellet was resuspended in 10 mL of buffer (PBS, NaCl 0.3 M, and Imidazole 5 mM, pH 7) and disrupted by sonication. The cell lysate was centrifuged at 13,000×g, 4 °C, for 20 min and the supernatant used for ChOA purification via affinity chromatography.
Purification of recombinant ChOA
Recombinant ChOA containing N-terminal His tag was purified from the soluble crude extract using nickel affinity chromatography (Ni-CAM HC Resin). The column (2 mL) was equilibrated with 30 mL of equilibration buffer (PBS, Imidazole 5 mM, NaCl 0.3 M; pH 7) at 1 mL/min. The supernatant was loaded onto the column and the column was washed with equilibrium buffer until the absorbance at 280 nm reached the basal level. To elute the protein, elution buffer (PBS, NaCl 0.3 M, and Imidazole 200 mM; pH 7) was used, and the released proteins were fractionated. The purity of the fractionated samples was evaluated by SDS-PAGE 12%. The pure fractions were pooled together and dialyzed against 50 mM sodium phosphate buffer at 4 °C, pH 7 for 16 h. Enzyme activity and protein concentration of the crude extract, flow-through, and pure enzyme were determined using the enzyme activity assay and Bradford protein assay (Aminian et al. 2013) and the resulting data used for determining purification yield and specific activity of recombinant ChOA.
Kinetic characterization of purified ChOA
The optimum pH for the recombinant enzyme activity was determined by the enzyme activity assay at 30 °C under various pH (3–11) conditions. The buffer systems were prepared according to Doukyu et al. (Doukyu et al. 2008). The recombinant ChOA activity was also assayed at different temperatures (30 °C–80 °C) in order to determine the recombinant enzyme optimum thermal activity. The Km and Vmax values for cholesterol were estimated from Lineweaver–Burk plots of data obtained with the assay solution containing 0–1 mM cholesterol.
Optimization of recombinant ChOA expression
Optimal host strain for ChOA expression
Evaluation of the E. coli host strains productivity for producing recombinant ChOA was performed by transformation of pET24-ChOA plasmid into BL21(DE3), BL21(DE3)pLysS, and Rosetta-gami2(DE3). The host strains were simultaneously induced with 0.5 mM IPTG and the protein expression continued for 6 h at 37 °C, 160 rpm. Following sonication, the cell lysates were centrifuged to remove insoluble materials, and the resulting supernatants were collected to determine the units of enzyme produced per liter of the culture media. The BL21(DE3)pLysS cells yielded the maximum level of active recombinant ChOA with 14 U/L activity (Fig. 1). In addition, the total amount of active enzyme obtained from Rosetta-gami2(DE3) (6.8 U/L) was higher than that of BL21(DE3) (3.2 U/L).
Optimal culture media for ChOA expression
To achieve the optimum production of soluble ChOA in BL21(DE3)pLysS, three different culture media (LB, TB, and SB) were evaluated. To compare the effect of the different culture media, overnight culture of BL21(DE3)pLysS containing pET24-ChOA was developed in LB at 37 °C. Pre-culture inoculum (1%) was transformed into freshly prepared LB, TB, and SB media and incubated at 37 °C until OD600nm reached 0.6. Subsequently, the cultures were induced with 0.5 mM IPTG and were grown for another 6 h at 37 °C, 160 rpm. Comparison of cell density and total enzyme activity in different media is demonstrated in Fig. 2. The highest biomass accumulation (OD600nm = 3.9) was achieved by TB medium. Also, ChOA assay indicated that recombinant protein productivity increased in TB medium when compared to the other media.
Pre-induction growth optimization
In order to determine the optimum induction time, four shake flasks were examined in parallel, with each culture induced at different growth phases. Cultures were induced with 0.5 mM IPTG when the OD600nm reached 0.3, 0.6, 1.2, and 1.8, representing early exponential, mid-exponential, late exponential, and stationary phases, respectively. The results, depicted in Fig. 3, indicate that ChOA yield was maximized to 28.8 U/L when induction was made at the mid-exponential growth phase (OD600nm = 0.6).
Inducer concentration optimization
Further, the effect of IPTG concentrations (0.05, 0.1, 0.25, 0.5, 1 mM) on ChOA productivity was investigated under the best conditions achieved so far (BL21(DE3)pLysS, TB medium, induction at OD600nm = 0.6). Figure 4 presents the results obtained by the performed experiments. As IPTG gradually increased up to 0.25 mM, the productivity also increased in the same way. Nevertheless, beyond 0.25 mM, reduction in the active enzyme yield was observed.
Optimal induction temperature and post-induction incubation time
To determine the optimal induction temperature and post-induction incubation time, three flasks containing TB media were cultivated under previously optimized conditions. After addition of IPTG (0.25 mM), the flasks were incubated at 15 °C, 25 °C, and 37 °C, separately. During incubation, 2 mL of culture was withdrawn from each flask at different time intervals (6, 8, 16, and 24 h). Enzyme activity assay revealed that recombinant ChOA production was markedly increased considerably when the induced culture medium was incubated at 15 °C for 24 h. As summarized in Fig. 5, the cell density and total enzyme activity decreased when the cultures were incubated at 37 °C even for 16 or 24 h.
Large-scale enzyme production
All the results above were obtained from small-scale cultures. Collecting all the optimal conditions together, we performed 500 mL culture of BL21(DE3)pLysS-pET24-ChOA at 15 °C for 24 h by induction with 0.25 mM IPTG at the mid-exponential phase (OD600nm ≃ 0.6). After sonication and obtaining a clarified crude extract by centrifugation, the total enzyme activity and total protein concentration were measured. As shown in Table 1, the total enzyme activity and total protein content were 78.5 U and 63 mg, respectively.
Purification of recombinant ChOA
The recombinant ChOA protein containing N-terminal 6 × His-Tag was purified by nickel column affinity chromatography. The pre-column, flow-through, and eluted fractions were analyzed by SDS-PAGE, with the results indicating that pure ChOA was efficiently eluted by 200 mM imidazole. As displayed in Fig. 6, lanes 5–9, ChOA was highly purified. Eluted fractions containing pure ChOA were pooled and dialyzed against 50 mM of sodium phosphate buffer at pH 7. Table 1 summarizes the data of purification steps. The overall yield of 86% and the approximately 5.7-fold increase in the overall purification were achieved by Ni-CAM affinity chromatography.
Properties of the purified cholesterol oxidase
The ChOA activity was measured at different values of pH and temperature. The enzyme activity retained more than 95% of its maximal activity within the pH range of 6–7 at 25 °C (Fig. 7a). The optimum temperature for the ChOA activity was determined, with the results indicating that the optimal temperature for ChOA activity was 60 °C (Fig. 7b). Further, the enzyme retained more than 60% of its activity at the temperatures from 40 °C to 70 °C under the test conditions. In addition, the enzyme had a relatively low activity at 30 °C (42%) and 80 °C (24%) compared to 60 °C. To calculate the Km and Vmax values of purified ChOA, the activity of the enzyme was assayed with a range of cholesterol concentrations (0–1 mM) at 25 °C, 0.1 M of potassium phosphate buffer pH 7. For Km and Vmax estimation, 1/V was plotted against 1/[S] in a Lineweaver–Burk plot (Fig. 8). Results indicated that the Km and Vmax values were found to be 13 µM and 7.2 µmol min−1 mg−1 respectively.
Cholesterol oxidase as a bacterial flavoenzyme has a great commercial value with a wide range of applications in various fields (Kumari and Kanwar 2012). In light of this, the most efficient production of the enzyme is desired in a recombinant form. There are several obstacles against the heterologous protein expression which results in the production of a recombinant protein at a very low or zero level. One of the simplest ways to address these issues is selecting a suitable host strain and optimizing the expression conditions (Rosano and Ceccarelli 2014). In the current study, several parameters were selected for optimization of the cholesterol oxidase production.
In the first step, three different E. coli hosts were used to produce recombinant ChOA. Among them, BL21(DE3)pLysS expressed relatively high levels of the active enzyme. pET expression system based on T7 promoter was used for efficient expression of our desired gene. High transcription rate is the advantage of this system but in some cases, this can lead to accumulation of misfolded proteins in inclusion body due to saturation of protein folding machinery (Bahreini et al. 2014). BL21(DE3)pLysS was designed to resolve this problem. In this way, pLyS plasmid consistently produces phage T7 lysozyme which can bind to T7 RNA polymerase and partially prevents the transcription of the recombinant gene that is under the control of T7 promoter (Stano and Patel 2004).
Culture media should be accurately selected given their effect on cell growth and metabolism. Therefore, the yield of protein expression may be affected by culture media composition (Sivashanmugam et al. 2009). In this regard, we performed our experiments using three different media consisting of LB, SB, and TB. We found that cholesterol oxidase productivity in TB media increased approximately by three times in comparison with LB media. High concentrations of yeast extract, superior buffering capacity, and the use of glycerol as the carbon source supplement enable high biomass accumulation and high ChOA production (Collins et al. 2013).
Bacterial growth phase at the time of induction as well as inducer concentration also affect the production of recombinant proteins (Ahmad et al. 2018). Accordingly, the effects of these parameters on our target protein yield were next examined individually. Figure 3 indicates that the productivity of the enzyme did not change significantly when IPTG was added during the entire exponential phase. However, the expression level decreased when induction was made at the stationary growth phase. Evaluation of biomass production during different induction times revealed that the addition of IPTG at the early exponential phase reduced biomass production; in return IPTG addition at the stationary phase led to increased biomass accumulation. When induction was made at the early exponential growth phase, the bacterial metabolic resources were channeled to producing recombinant protein constituting 50% of the total cellular protein (Jevševar et al. 2005; Jin et al. 2012). Based on this reasoning, we should expect lowered cellular growth rate following the early exponential phase induction. Our experiment also showed that great production of recombinant ChOA was obtained when IPTG concentration was 0.25 mM.
Several studies have suggested that post-induction temperature as well as incubation time can affect the activity and yield of recombinant protein production (Caspeta et al. 2009; Khow and Suntrarachun 2012; Sahdev et al. 2008; Saïda 2007). In addition, Mizukami et al. have reported that different expression temperatures finally led to equal-mass production of the recombinant enzyme with different total activity. They suggested that in the cells cultured at a lower temperature the recombinant enzyme seems to exist as an active form, while as a rather denatured form in the cells cultured at a higher temperature (Mizukami et al. 1986). In light of these findings, we also investigated the effect of different post-induction temperatures (15 °C, 25 °C, and 37 °C) along with post-induction incubation times (6, 8, 16, and 24) on the yield of recombinant ChOA. As can be seen clearly in Fig. 5, reducing temperature down to 15 °C together with extending the incubation period up to 24 h enhanced the enzyme productivity by approximately 7.5 times relative to the same condition at 37 °C. Generally, metabolic burden usually occurs in recombinant bacteria (Bentley et al. 1990). Accordingly, high-rate produced recombinant proteins may accumulate in insoluble aggregates (inclusion body) as a direct consequence of overwhelming the host folding machinery (Sørensen and Mortensen 2005). In addition, hydrophobic interactions which are a key factor in the formation of inclusion bodies would decline if temperature is lowered (Kiefhaber et al. 1991; Löw et al. 2012; Ma et al. 2013).
Furthermore, in order to study the enzymatic characteristics of the recombinant ChOA, large-scale production of ChOA was performed under the optimized conditions. Maximum yield of recombinant ChOA production was determined to be 1.25 U/mg. Nomura et al. produced ChOA by Streptomyces sp. SA-COO and E. coli JM109. They achieved 0.69 U/mg ChOA when cholesterol oxidase was produced by Streptomyces sp. SA-COO. Further, they obtained 1.5 U/mg recombinant enzyme when N-terminal modified ChOA was expressed in E. coli JM109. The characterization of purified recombinant ChOA indicated that the recombinant enzyme was most active at 50 °C–70 °C, with 60 °C being the optimum temperature, which is the same as that of other Streptomycetes (Lartillot and Kedziora 1990; Nishiya et al. 1997; Tabatabaei Yazdi et al. 2001; Tomioka et al. 1976). However, the enzyme retained only 24% of its activity at 80 °C. Furthermore, activity assay at different pH values revealed that the optimum pH for enzyme activity was 7. Most reports have demonstrated the optimum pH for cholesterol oxidase from other Streptomycetes as about 6.5–8 (Kamei et al. 1978; Lartillot and Kedztora 1990; Smith and Brooks 1976). The Km value for cholesterol was calculated to be 13 µM for purified ChOA. This value is consistent with the study of Nishiya et al. (Nishiya et al. 1997), which is lower than that of the enzymes from S. hygroscopicus and S. virginiae (Gadda et al. 1997; Li et al. 2010).
In conclusion, the results of our study suggested that optimization of ChOA expression conditions in E. coli significantly enhanced the enzyme productivity by approximately 50 times. The affinity purified ChOA retained the enzyme characteristics as reported previously.
Ahmad I, Nawaz N, Darwesh NM, Ur Rahman S, Mustafa MZ, Khan SB, Patching SG (2018) Overcoming challenges for amplified expression of recombinant proteins using Escherichia coli. Protein Expr Purif 144:12–18. https://doi.org/10.1016/j.pep.2017.11.005
Allain CC, Poon LS, Chan CS, Richmond W, Fu PC (1974) Enzymatic determination of total serum cholesterol. Clin Chem 20(4):470–475
Aminian M, Nabatchian F, Vaisi-Raygani A, Torabi M (2013) Mechanism of Coomassie Brilliant Blue G-250 binding to cetyltrimethylammonium bromide: an interference with the Bradford assay. Anal Biochem 434(2):287–291
Bahreini E, Aghaiypour K, Abbasalipourkabir R, Goodarzi MT, Saidijam M, Safavieh SS (2014) An optimized protocol for overproduction of recombinant protein expression in Escherichia coli. Prep Biochem Biotechnol 44(5):510–528. https://doi.org/10.1080/10826068.2013.833116
Bentley WE, Mirjalili N, Andersen DC, Davis RH, Kompala DS (1990) Plasmid-encoded protein: the principal factor in the “metabolic burden” associated with recombinant bacteria. Biotechnol Bioeng 35(7):668–681. https://doi.org/10.1002/bit.260350704
Brigidi P, Bolognani F, Rossi M, Cerre C, Matteuzzi D (1993) Cloning of the gene for cholesterol oxidase in Bacillus spp., Lactobacillus reuteri and its expression in Escherichia coli. Lett Appl Microbiol 17(2):61–64. https://doi.org/10.1111/j.1472-765x.1993.tb00371.x
Caspeta L, Flores N, Pérez NO, Bolívar F, Ramírez OT (2009) The effect of heating rate on Escherichia coli metabolism, physiological stress, transcriptional response, and production of temperature-induced recombinant protein: a scale-down study. Biotechnol Bioeng 102(2):468–482. https://doi.org/10.1002/bit.22084
Cho H-J, Choi K-P, Yamashita M, Morikawa H, Murooka Y (1995) Introduction and expression of the Streptomyces cholesterol oxidase gene (ChoA), a potent insecticidal protein active against boll weevil larvae, into tobacco cells. Appl Microbiol Biotechnol 44(1–2):133–138
Collins T, Azevedo-Silva J, da Costa A, Branca F, Machado R, Casal M (2013) Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation. Microbial Cell Fact 12(1):21. https://doi.org/10.1186/1475-2859-12-21
Corbin DR, Greenplate JT, Wong EY, Purcell JP (1994) Cloning of an insecticidal cholesterol oxidase gene and its expression in bacteria and in plant protoplasts. Appl Environ Microbiol 60(12):4239–4244
Doukyu N (2009) Characteristics and biotechnological applications of microbial cholesterol oxidases. Appl Microbiol Biotechnol 83(5):825–837. https://doi.org/10.1007/s00253-009-2059-8
Doukyu N, Shibata K, Ogino H, Sagermann M (2008) Purification and characterization of Chromobacterium sp. DS-1 cholesterol oxidase with thermal, organic solvent, and detergent tolerance. Appl Microbiol Biotechnol 80(1):59
Doukyu N, Shibata K, Ogino H, Sagermann M (2009) Cloning, sequence analysis, and expression of a gene encoding Chromobacterium sp. DS-1 cholesterol oxidase. Appl Microbiol Biotechnol 82(3):479–490. https://doi.org/10.1007/s00253-008-1775-9
Fujishiro K, Ota T, Hasegawa M, Yamaguchi K, Mizukami T, Uwajima T (1990) Isolation and identification of the gene of cholesterol oxidase from Brevibacterium sterolicum ATCC 21387, a widely used enzyme in clinical analysis. Biochem Biophys Res Commun 172(2):721–727. https://doi.org/10.1016/0006-291X(90)90734-5
Gadda G, Wels G, Pollegioni L, Zucchelli S, Ambrosius D, Pilone MS, Ghisla S (1997) Characterization of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum. Eur J Biochem 250(2):369–376
Horii M, Ishizaki T, Paik SY, Manome T, Murooka Y (1990) An operon containing the genes for cholesterol oxidase and a cytochrome P-450-like protein from a Streptomyces sp. J Bacteriol 172(7):3644–3653. https://doi.org/10.1128/jb.172.7.3644-3653.1990
Ishizaki T, Hirayama N, Shinkawa H, Nimi O, Murooka Y (1989) Nucleotide sequence of the gene for cholesterol oxidase from a Streptomyces sp. J Bacteriol 171(1):596–601
Jevševar S, Gaberc-Porekar V, Fonda I, Podobnik B, Grdadolnik J, Menart V (2005) Production of nonclassical inclusion bodies from which correctly folded protein can be extracted. Biotechnol Prog 21(2):632–639. https://doi.org/10.1021/bp%5b0497839
Jin DJ, Cagliero C, Zhou YN (2012) Growth rate regulation in Escherichia coli. FEMS Microbiol Rev 36(2):269–287. https://doi.org/10.1111/j.1574-6976.2011.00279.x
Kamei T, Takiguchi Y, Suzuki H, Matsuzaki M, Nakamura S (1978) Purification of 3β-hydroxysteroid oxidase of Streptomyces violascens origin by affinity chromatography on cholesterol. Chem Pharm Bull 26(9):2799–2804
Khow O, Suntrarachun S (2012) Strategies for production of active eukaryotic proteins in bacterial expression system. Asian Pac J Trop Biomed 2(2):159. https://doi.org/10.1016/S2221-1691(11)60213-X
Kiefhaber T, Rudolph R, Kohler H-H, Buchner J (1991) Protein aggregation in vitro and in vivo: a quantitative model of the kinetic competition between folding and aggregation. Nat Biotechnol 9(9):825
Kumari L, Kanwar SS (2012) Cholesterol oxidase and its applications. Adv Microbiol 2(2):49–65
Lartillot S, Kedziora P (1990) Production, purification and some properties of cholesterol oxidase from a streptomyces Sp. Prep Biochem 20(1):51–62
Li B, Wang W, Wang FQ, Wei DZ (2010) Cholesterol oxidase ChoL is a critical enzyme that catalyzes the conversion of diosgenin to 4-ene-3-keto steroids in Streptomyces virginiae IBL-14. Appl Microbiol Biotechnol 85(6):1831–1838. https://doi.org/10.1007/s00253-009-2188-0
Liu WH, Meng MH, Chen KS (1988) Purification and some properties of cholesterol oxidases produced by an inducible and a constitutive mutant of Arthrobacter simplex. Agric Biol Chem 52(2):413–418. https://doi.org/10.1271/bbb1961.52.413
Löw C, Jegerschöld C, Kovermann M, Moberg P, Nordlund P (2012) Optimisation of over-expression in E. coli and biophysical characterisation of human membrane protein synaptogyrin 1. PLoS ONE 7(6):e38244. https://doi.org/10.1371/journal.pone.0038244
Lv C, Tang Y, Wang L, Ji W, Chen Y, Yang S, Wang W (2002) Bioconversion of yolk cholesterol by extracellular cholesterol oxidase from Brevibacterium sp. Food Chem 77(4):457–463. https://doi.org/10.1016/S0308-8146(01)00381-8
Ma P, Varela F, Magoch M, Silva AR, Rosario AL, Brito J, Oliveira TF, Nogly P, Pessanha M, Stelter M, Kletzin A, Henderson PJ, Archer M (2013) An efficient strategy for small-scale screening and production of archaeal membrane transport proteins in Escherichia coli. PLoS ONE 8(10):e76913. https://doi.org/10.1371/journal.pone.0076913
MacLachlan J, Wotherspoon A, Ansell R, Brooks C (2000) Cholesterol oxidase: sources, physical properties and analytical applications. J Steroid Biochem Mol Biol 72(5):169–195
Mizukami T, Komatsu Y, Hosoi N, Itoh S, Oka T (1986) Production of active human interferon-β in E. coli I. Preferential production by lower culture temperature. Biotechnology letters 8(9):605–610
Molnár I, Choi KP, Hayashi N, Murooka Y (1991) Secretory overproduction of Streptomyces cholesterol oxidase by Streptomyces lividans with a multi-copy shuttle vector. J Ferment Bioeng 72(5):368–372. https://doi.org/10.1016/0922-338X(91)90089-Y
Moradpour Z, Ghasemian A (2016) Protein engineering of microbial cholesterol oxidases: a molecular approach toward development of new enzymes with new properties. Appl Microbiol Biotechnol 100(10):4323–4336. https://doi.org/10.1007/s00253-016-7497-5
Murooka Y, Ishizaki T, Nimi O, Maekawa N (1986) Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ702. Appl Environ Microbiol 52(6):1382–1385
Nishiya Y, Harada N, Teshima SI, Yamashita M, Fujii I, Hirayama N, Murooka Y (1997) Improvement of thermal stability of Streptomyces cholesterol oxidase by random mutagenesis and a structural interpretation. Protein Eng 10(3):231–235
Nomura N, Choi KP, Yamashita M, Yamamoto H, Murooka Y (1995) Genetic modification of the Streptomyces cholesterol oxidase gene for expression in Escherichia coli and development of promoter-probe vectors for use in enteric bacteria. J Ferment Bioeng 79(5):410–416. https://doi.org/10.1016/0922-338X(95)91253-2
Ohta T, Fujishiro K, Yamaguchi K, Uwajima T, Aisaka K, Hasegawa M (1992) Hyperexpression and analysis of choB encoding cholesterol oxidase of Bvevibactevium sterolicum in Escherichia coli and Streptomyces lividans. Biosci Biotechnol Biochem 56(11):1786–1791. https://doi.org/10.1271/bbb.56.1786
Pollegioni L, Piubelli L, Molla G (2009) Cholesterol oxidase: biotechnological applications. FEBS J 276(23):6857–6870. https://doi.org/10.1111/j.1742-4658.2009.07379.x
Purcell JP, Greenplate JT, Jennings MC, Ryerse JS, Pershing JC, Sims SR, Prinsen MJ, Corbin DR, Tran M, Sammons RD, Stonard RJ (1993) Cholesterol oxidase: a potent insecticidal protein active against boll weevil larvae. Biochem Biophys Res Commun 196(3):1406–1413. https://doi.org/10.1006/bbrc.1993.2409
Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172. https://doi.org/10.3389/fmicb.2014.00172
Sahdev S, Khattar SK, Saini KS (2008) Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem 307(1–2):249–264. https://doi.org/10.1007/s11010-007-9603-6
Saïda F (2007) Overview on the expression of toxic gene products in Escherichia coli. Curr Protoc Protein Sci 50(1):5.19. 1–5.19. 13. https://doi.org/10.1002/0471140864.ps0519s50
Serajzadeh S, Alemzadeh I (2010) Low-cholesterol milk production using cholesterol oxidase from Nocardia erythropolis. Aust J Dairy Technol 65(1):10–14
Sivashanmugam A, Murray V, Cui C, Zhang Y, Wang J, Li Q (2009) Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein Sci 18(5):936–948
Smith AG, Brooks CJ (1976) Cholesterol oxidases: properties and applications. J Steroid Biochem 7(9):705–713
Smith M, Sullivan C, Goodman N (1991) Reactivity of milk cholesterol with bacterial cholesterol oxidases. J Agric Food Chem 39(12):2158–2162
Solaiman DKY, Somkuti GA (1991) Expression of streptomycete cholesterol oxidase in Escherichia coli. J Ind Microbiol 8(4):253–258. https://doi.org/10.1007/BF01576063
Solaiman DKY, Somkuti GA (1995) Expression of Streptomyces melC and choA genes by a cloned Streptococcus thermophilus promoter STP2201. J Ind Microbiol 15(1):39–44. https://doi.org/10.1007/BF01570011
Solaiman DKY, Somkuti GA, Steinberg DH (1992) Construction and characterization of shuttle plasmids for lactic acid bacteria and Escherichia coli. Plasmid 28(1):25–36. https://doi.org/10.1016/0147-619X(92)90033-7
Somkuti GA, Solaiman DKY (1997) ST(P2201), a chromosomal promoter sequence of Streptococcus thermophilus. Curr Microbiol 35(3):180–185. https://doi.org/10.1007/s002849900235
Somkuti G, Solaiman D, Johnson T, Steinberg D (1991) Transfer and expression of a Streptomyces cholesterol oxidase gene in Streptococcus thermophilus. Biotechnol Appl Biochem 13(2):238–245. https://doi.org/10.1111/j.1470-8744.1991.tb00153.x
Somkuti GA, Solaiman DKY, Steinberg DH (1995) Native promoter-plasmid vector system for heterologous cholesterol oxidase synthesis in streptococcus thermophilus. Plasmid 33(1):7–14. https://doi.org/10.1006/plas.1995.1002
Sørensen HP, Mortensen KK (2005) Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Fact 4(1):1. https://doi.org/10.1186/1475-2859-4-1
Stano NM, Patel SS (2004) T7 lysozyme represses T7 RNA polymerase transcription by destabilizing the open complex during initiation. J Biol Chem 279(16):16136–16143. https://doi.org/10.1074/jbc.M400139200
Tabatabaei Yazdi M, Malekzadeh F, Zarrini G, Faramarzi MA, Kamranpour N, Khaleghparast S (2001) Production of cholesterol oxidase by a newly isolated Rhodococcus sp. World J Microbiol Biotechnol 17(7):731–737. https://doi.org/10.1023/A:1012993532686
Tomioka H, Kagawa M, Nakamura S (1976) Some enzymatic properties of 3β-hydroxysteroid oxidase produced by Streptomyces violascens. J Biochem 79(5):903–915
Vrielink A, Ghisla S (2009) Cholesterol oxidase: biochemistry and structural features. FEBS J 276(23):6826–6843. https://doi.org/10.1111/j.1742-4658.2009.07377.x
Yehia HM, Hassanein WA, Ibraheim SM (2015) Purification and characterisation of the extracellular cholesterol oxidase enzyme from Enterococcus hirae. BMC Microbiol 15(1):178. https://doi.org/10.1186/s12866-015-0517-2
AF performed experiments and wrote the manuscript. AG designed experiments. ML and SV helped with the experimentation. MA designed and directed experiments, analyzed data and revised the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Consent for publication
Provided during submission process. Not applicable.
Compliance with ethical standards
This study was reviewed and approved by the Ethics Committee of the Tehran University of Medical Sciences (IR.TUMS.REC.1395.2376).
This study was funded by Tehran University of Medical Sciences (Grant Number 30859).
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Fazaeli, A., Golestani, A., Lakzaei, M. et al. Expression optimization of recombinant cholesterol oxidase in Escherichia coli and its purification and characterization. AMB Expr 8, 183 (2018). https://doi.org/10.1186/s13568-018-0711-3