- Original article
- Open Access
A two-stage temperature control strategy enhances extracellular secretion of recombinant α-cyclodextrin glucosyltransferase in Escherichia coli
© The Author(s) 2017
- Received: 7 June 2017
- Accepted: 17 August 2017
- Published: 23 August 2017
The effects of temperature on extracellular secretion of the α-cyclodextrin glucosyltransferase (α-CGTase) from Paenibacillus macerans JFB05-01 by Escherichia coli were investigated. When protein expression was induced at constant temperature, the greatest amount of extracellular recombinant α-CGTase was produced at 25 °C. Higher or lower induction temperatures were not conducive to extracellular secretion of recombinant α-CGTase. To enhance extracellular secretion of α-CGTase by E. coli, a two-stage temperature control strategy was adopted. When expression was induced at 25 °C for 32 h, and then the temperature was shifted to 30 °C, the extracellular α-CGTase activity at 90 h was 45% higher than that observed when induction was performed at a constant temperature of 25 °C. Further experiments suggested that raising the induction temperature can benefit the transport of recombinant enzyme and compensate for the decreased rate of recombinant enzyme synthesis during the later stage of expression. This report provides a new method of optimizing the secretory expression of recombinant enzymes by E. coli.
- Temperature control
- Extracellular secretion
- Recombinant enzymes
- E. coli
The cyclic oligosaccharides α-, β-, and γ-cyclodextrin consist of 6, 7, and 8 glucose units, respectively, linked by α-1, 4-glycosidic bonds. Cyclodextrins form inclusion complexes with many different small, hydrophobic guest molecules, improving their solubility and stability in aqueous environments. This property makes it have many applications in scientific, medical and industrial fields (Roy et al. 2017). The industrial use of α-cyclodextrin is in its infancy, yet is still expanding because of its small internal cavity, high water solubility, and resistance to enzymatic hydrolysis. Previous reports have shown that α-cyclodextrin can be used as a carrier of active ingredients, a solubilizer of lipids, a stabilizer of oils, a modifier of flavors or aromas, and a natural soluble dietary fiber (Aytac and Uyar 2016; Li et al. 2010b, 2014a).
With the expanding use of cyclodextrins on an industrial scale, the cyclodextrin glucosyltransferases (CGTases, EC 188.8.131.52), which catalyze the formation of cyclodextrins, have received increased scientific interest. Although CGTases can be obtained from a wide range of bacteria, the characteristics of the CGTases from Bacillus strains are among the closest to industrial requirements (Tonkova 1998). Early work focused on CGTase production in Bacillus strains (Gawande et al. 1998; Rosso et al. 2002), and efforts were made to improve CGTase yield by manipulating environmental factors (Arce-Vazquez et al. 2016; Es et al. 2016). Unfortunately, the strict regulatory mechanisms present in wild-type strains have limited productivity enhancements, resulting in high costs and low yields.
A substantial improvement in CGTase expression was observed when the overexpression was performed in recombinant Escherichia coli (Mana et al. 2015; Sonnendecker et al. 2017). Unfortunately, previous reports have demonstrated that the CGTases expressed in E. coli usually accumulated in the cytosol as biologically inactive inclusion bodies (Makrides 1996; Choi and Lee 2004), and the refolding processes have been proved to be inconvenient (Li et al. 2005). Although secretion into the periplasm is helpful for the rapid isolation of recombinant proteins, current methods for the selective release of periplasmic proteins are not suitable for large-scale production (Yang et al. 1998; Jeang et al. 2005). Therefore, the limitations of cytosolic and periplasmic expression of CGTase make the extracellular secretion of CGTases highly needed.
In our previous study, the α-CGTase gene from Paenibacillus macerans JFB05-01 was cloned into the plasmid vector pET-20b(+). This plasmid was then inserted into E. coli BL21(DE3) to form a strain used for the extracellular expression of α-CGTase by E. coli (Li et al. 2010a, b). The greatest amount of extracellular recombinant α-CGTase was produced when expression was induced at a constant temperature of 25 °C (Li et al. 2010a, b). Extracellular α-CGTase secretion was inhibited when expression was induced at temperatures >30 °C, and very little recombinant enzyme was obtained at 37 °C (Li et al. 2010a, b). Additional studies were devoted to improving the yields of these recombinant α-CGTase by optimizing the composition of the culture medium (Ding et al. 2010; Li et al. 2013a, b). When a one-stage temperature control strategy was used, the membrane permeability was generally at a low level. This low degree of membrane permeability did not favor the secretion of mature α-CGTase into the culture medium. Using a variable temperature control strategy, the membrane permeability may be increased. Therefore, in this study, a novel two-stage temperature control strategy was developed to further improve extracellular expression of P. macerans α-CGTase by E. coli. The underlying mechanisms for the enhanced enzyme secretion are discussed.
Expression plasmid and chemicals
Construction of the recombinant plasmid cgt/pET-20b(+), which directs expression of the wild-type α-CGTase from P. macerans strain JFB05-01 (CCTCC M203062) fused to the pelB signal peptide, has been described in a previous report (Li et al. 2009). Peptone and yeast extract powder were obtained from Oxoid (Basingstoke, Hampshire, United Kingdom). Isopropyl β-d-1-thiogalactopyranoside (IPTG), O-nitrophenyl-β-d-galactopyranoside (ONPG) and N-phenyl-α-naphthylamine (NPN) were purchased from Beyotime Institute of Biotechnology (Nantong, China). Glycerin and methyl orange were purchased from Shanghai Chemical Reagent Ltd. (Shanghai, China). All inorganic compounds were of reagent grade or higher quality.
A single colony of E. coli BL21 (DE3) harboring plasmid cgt/pET-20b(+) was used to inoculate 50 mL of Luria–Bertani (LB) medium supplemented with 100 mg/mL ampicillin (inoculum size, approximately 0.1%). This starter culture was incubated on a rotary shaker (200 rpm) at 37 °C until the optical density at 600 nm (OD600) reached 0.6 (about 8 h). The resulting culture was diluted (1:25) into 100 mL of terrific broth medium in a 500-mL flask, and IPTG was added to a final concentration of 0.01 mM to induce protein expression. The induction was allowed to proceed on a rotary shaker (200 rpm) at the specified temperature for 90 h. Samples of the culture were taken at intervals and analyzed for cell concentration and enzyme activity.
Cell fractionation was performed as previously described, with minor modifications (Li et al. 2010a, b). A 1-mL sample of the culture solution was centrifuged at 10,000 rpm for 10 min and the supernatant was collected. To separate the periplasmic fraction, the bacterial pellet from the 1-mL sample was washed twice with pure water and then completely resuspended in pure water containing 25% (w/v) sucrose and 1 mM EDTA. This suspension was adjusted to a final volume of 1 L, incubated on ice for 2 h, and then centrifuged at 10,000 rpm for 5 min. The supernatant was collected as the periplasmic fraction. The pellet was resuspended in l mL of 10 mM sodium phosphate buffer (pH 6.2) containing 0.5 mM calcium chloride and disrupted by ultrasonication with a sonifier (Branson, USA) for 5 min. After centrifugation at 10,000 rpm for 10 min, the residual cell fragments were mixed with 100 μL of 1% (w/v) SDS-PAGE loading buffer and heated for 10 min in a boiling water bath. After a final centrifugation, the α-CGTase inclusion bodies were in the upper buffer.
α-CGTase activity assay
α-CGTase activity was determined using the methyl orange method (Li et al. 2013a, b). The culture supernatant (0.1 mL) was mixed with 0.9 mL of 5% (w/v) soluble starch in 50 mM phosphate buffer (pH 6.0) and incubated at 40 °C for 10 min. After terminating the reaction by the addition of 1.0 mL HCl (1.0 M), 1.0 mL of 0.1 mM methyl orange in 50 mM phosphate buffer (pH 6.0) was added. After the mixture had reacted at 16 °C for 20 min, the amount of α-cyclodextrin in the mixture was determined by measuring the absorbance at 505 nm. One unit of α-cyclodextrin-forming activity was defined as the amount of enzyme able to produce 1 µmol of cyclodextrin per min.
Analysis of inner and outer membrane permeability
Samples were removed from the fermentation specified times after induction and centrifuged at 10,000 rpm for 10 min. The cell pellets were washed twice with 10 mM sodium phosphate buffer (pH 7.4) and diluted with the same buffer until the OD600 reached 0.5. Samples (1 mL) of these cell suspensions were used to assess the permeability of their inner and outer membranes as described below.
A previously described absorbance assay was used to evaluate the permeability of the inner membrane (Liao et al. 2004). Briefly, cell samples described above were mixed with ONPG (100 μg/mL) to assess permeability of the inner membrane. Cleavage of the ONPG that entered the cell, which is catalyzed by cytosolic β-galactosidase, was determined by measuring the absorption of light at 420 nm using a spectrophotometer. Measurements were taken every 5 or 10 min for 2 h.
Completeness of the outer membrane was assessed using a previously described NPN fluorescence assay (Eriksson et al. 2002). The fluorescence of NPN is weaker in aqueous solution than it is in hydrophobic environments. When NPN is applied to intact cells, it is excluded from the cells’ interior by the lipopolysaccharide layer of the cells’ outer membranes. Once the outer membrane is compromised, NPN gains access to the lipid bilayer and its fluorescence becomes strong in this hydrophobic environment. Cell suspensions (1 mL, prepared as described in the previous paragraph) were treated with NPN at a final concentration of 10 mM. Fluorescence was measured every 5 or 10 min for 2 h using excitation and emission wavelengths of 350 and 428 nm, respectively, and slit widths of 1 nm. Elevated NPN fluorescence was considered evidence of compromised cell membranes.
All measurements were performed in triplicate. The mean and standard deviations of the data collected were calculated using SPSS 17.0 software (SPSS Incorporated, Chicago, Illinois, USA).
A two-stage temperature control strategy enhanced extracellular α-CGTase secretion
Cell membrane permeability increased with time and temperature
Movement of α-CGTase between compartments
High initial induction temperature hinders the α-CGTase translocation
Extracellular α-CGTase production occurs in a series of steps. The pre-α-CGTase produced on the ribosome contains an N-terminal pelB signal peptide sequence. This N-terminal signal peptide directs translocation of the pre-α-CGTase across the inner membrane to the periplasmic space via the SecB pathway (Su et al. 2012). During this process, the signal peptide is removed. Once α-CGTase enters the periplasm, it has two potential fates: it can pass through the outer membrane and enter into the culture medium, or it can aggregate and form inclusion bodies in the periplasmic space (Li et al. 2014a, b).
A previous study showed that the greatest amount of extracellular α-CGTase was produced when the induction was conducted at a constant temperature of 25 °C (Li et al. 2010a, b). Extracellular α-CGTase production was inhibited when the induction temperatures was >30 °C, and very little recombinant enzyme could be obtained at 37 °C. The reason of this phenomenon was probably that the pre-α-CGTase formed inclusion bodies at the inner membrane at temperature above 30 °C, which could block the pre-protein translocation channels and suppress the entrance of newly synthesized pre-CGTase into the periplasm (Chen et al. 2014). At 25 °C, pre-α-CGTase synthesis proceeded at a desirable rate and most of the pre-CGTase passed smoothly through the inner membrane into the periplasm, and then folded correctly (Mergulhao et al. 2005; Fang et al. 2010). The rate of protein synthesis at 25 °C may have prevented the target protein from saturating the secretion machinery and have facilitated the translocation of α-CGTase across both E. coli membranes (Yamabhai et al. 2008; Fang et al. 2010).
A two-stage temperature control strategy enhanced the extracellular α-CGTase production
Having previously established that an initial induction temperature of 25 °C was optimal (Li et al. 2010a, b), we considered changes that would increase α-CGTase flux across both the inner and outer membranes without also increasing the rate of α-CGTase aggregation in the cytoplasm or periplasmic space. Increasing the temperature is a reasonable strategy to increase the α-CGTase movement across the membranes since it can increase the membrane permeability. However, prolonged induction periods at elevated temperature, especially at 37 °C, may cause cell lysis, which would decrease productivity and secretion capacity (Mana et al. 2015). Therefore, the second-stage temperatures between 30 and 37 °C were investigated during the total induction period of 90 h. The timing of the temperature shift had to be selected to minimize formation of inclusion bodies through accelerating translation. In the first experiment, 24 h was chosen because the inspection of the growth curve (Fig. 3) revealed that 24 h was well past the mid-point. The results showed that increasing the temperature increased the initial rate of α-CGTase production, but temperatures >30 °C gave poorer overall yields at 90 h of induction. This phenomenon was perhaps due to the premature cell lysis (Mana et al. 2015).
After selecting 30 °C as the optimal second-stage temperature, we decided to further investigate the timing of the temperature shift. The time course of α-CGTase activity in the periplasmic space during induction at 25 °C was clearly biphasic, with an early peak at 14 h of induction and a late peak at approximately 64 h (Fig. 5). We decided to investigate the temperature shift times beginning with the early peak (14 h, Fig. 5) and extending through late-log phase (36 h, Fig. 3). The optimal yield of the extracellular α-CGTase was finally obtained with a temperature shift time of 32 h (Fig. 5).
Enhanced membrane permeability increased α-CGTase production
The mechanistic data presented in Figs. 3, 4 and 5 strongly suggest that the two-stage induction strategy increased membrane permeability, which caused increased extracellular α-CGTase production. The increased inner membrane permeability in the ONPG study (Fig. 3) and the decreased amount of cytosolic protein and inclusion bodies in the SDS-PAGE study (Fig. 4) suggest that the two-stage induction strategy increased E. coli inner membrane permeability and then accelerated the transit of the α-CGTase across the inner membrane. This is confirmed by the increased α-CGTase activity that observed in the periplasmic space between 32 and 60 h of induction (Fig. 5). This accumulation of the α-CGTase activity in the periplasmic space further suggests that outer membrane permeability plays a significant role in the extracellular expression. The low degree of the outer membrane permeability was the main reason why only a small portion of the mature α-CGTase was secreted into the culture medium during the early stage of induction. The increased permeability of the outer membrane in the NPN study (Fig. 3) and the decreased amount of periplasmic α-CGTase in the SDS-PAGE study (Fig. 4) suggest that the two-stage induction strategy increased the E. coli outer membrane permeability, and then accelerated the transport of the α-CGTase across the outer membrane. This is confirmed by both the rapid decrease in the periplasmic α-CGTase activity during 60–90 h of induction and the simultaneous increase in the extracellular α-CGTase activity (Fig. 2).
In summary, this study showed that when the α-CGTase from P. macerans strain JFB05-01 was expressed in E. coli as a recombinant fusion protein carrying a pelB leader sequence, a two-stage induction temperature control strategy can help to obtain the optimal extracellular α-CGTase production. In this two-stage induction temperature control strategy, induction was conducted at 25 °C for 32 h, and then the temperature was shifted to 30 °C and the induction was continued for an additional 58 h. Using this two-stage induction control strategy, the extracellular α-CGTase activity was increased by 45% compared with the induction at a constant temperature of 25 °C. The primary mechanism responsible for the increase of the α-CGTase production was due to the increase of the membrane permeability. This is the first report describing a two-stage temperature control strategy used for increasing the extracellular α-CGTase production in E. coli.
- E. coli :
sodium dodecyl sulfate polyacrylamide gel electrophoresis
YL planned and carried out the experiments, analyzed the data and wrote the manuscript; JL, LS, YlW, BJL and RJ carried out the experiments; CML and ZBG reviewed the manuscript; ZFL participated in the data analysis and finalized the manuscript. All authors read and approved the final manuscript.
We thank the laboratory staff member of School of Food Science and Technology, Jiangnan University for all their support. We also thank Dr. Xiaofeng Ban for valuable guidance.
The authors declare that they have no competing interests.
Availability of data and materials
We conducted all the experiments and generated the data. All of the data are shown in the figures.
Ethics approval and consent to participant
Not applicable. This article does not contain any studies with human participants or animals.
This work was supported by Jiangnan University and the expenses were covered by Cereal and Starch Center.
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- Arce-Vazquez MB, Ponce-Alquicira E, Delgado-Fornué E, Pedroza-Islas R, Díaz-Godínez G, Soriano-Santos J (2016) Integral use of amaranth starch to obtain cyclodextrin glycosyltransferase, by Bacillus megaterium, to produce beta-cyclodextrin. Front Microbiol 7:11. doi:10.3389/fmicb.2016.01513 View ArticleGoogle Scholar
- Aytac Z, Uyar T (2016) Antioxidant activity and photostability of α-tocopherol/β-cyclodextrin inclusion complex encapsulated electrospun polycaprolactone nanofibers. Eur Polym J 79:140–149. doi:10.1016/j.eurpolymj.2016.04.029 View ArticleGoogle Scholar
- Chen WB, Nie Y, Xu Y, Xiao R (2014) Enhancement of extracellular pullulanase production from recombinant Escherichia coli by combined strategy involving auto-induction and temperature control. Bioprocess Biosyst Eng 37:601–608. doi:10.1007/s00449-013-1026-z View ArticlePubMedGoogle Scholar
- Choi JH, Lee SY (2004) Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 64:625–635. doi:10.1007/s00253-004-1559-9 View ArticlePubMedGoogle Scholar
- Ding R, Li Z, Chen S, Wu D, Wu J, Chen J (2010) Enhanced secretion of recombinant α-cyclodextrin glucosyltransferase from E. coli by medium additives. Prog Biotechnol 45:880–886. doi:10.1016/j.procbio.2010.02.009 Google Scholar
- Eriksson M, Nielsen PE, Good L (2002) Cell permeabilization and uptake of antisense peptide–peptide nucleic acid (PNA) into Escherichia coli. J Biol Chem 277:7144–7147. doi:10.1074/jbc.M106624200 View ArticlePubMedGoogle Scholar
- Es I, Ribeiro MC, dos Santos SR, Khaneghah AM, Rodriguez AG, Amaral AC (2016) Production of cyclodextrin glycosyltransferase by immobilized Bacillus sp. on chitosan matrix. Bioproc Biosyst Eng 39:1487–1500. doi:10.1007/s00449-016-1625-6 View ArticleGoogle Scholar
- Fang N, Zhong CQ, Liang X, Tang XF, Tang B (2010) Improvement of extracellular production of a thermophilic subtilase expressed in Escherichia coli by random mutagenesis of its N-terminal propeptide. Appl Microbiol Biotechnol 85:1473–1481. doi:10.1007/s00253-009-2183-5 View ArticlePubMedGoogle Scholar
- Gawande BN, Singh RK, Chauhan AK, Goel A, Patkar AY (1998) Optimization of cyclomaltodextrin glucanotransferase production from Bacillus firmus. Enzyme Microb Technol 22:288–291. doi:10.1016/s0141-0229(97)00184-1 View ArticleGoogle Scholar
- Jeang CL, Lin DG, Hsieh SH (2005) Characterization of cyclodextrin glycosyltransferase of the same gene expressed from Bacillus macerans, Bacillus subtilis, and Escherichia coli. J Agric Food Chem 53:6301–6304. doi:10.1021/jf0503356 View ArticlePubMedGoogle Scholar
- Li SG, Kweon DH, Lee DH, Park YC, Seo JH (2005) Coexpression of folding accessory proteins for production of active cyclodextrin glycosyltransferase of Bacillus macerans in recombinant Escherichia coli. Protein Expr Purif 41:426–432. doi:10.1016/j.pep.2005.01.017 View ArticleGoogle Scholar
- Li ZF, Zhang JY, Wang M, Gu ZB, Du GC, Li JK, Wu J, Chen J (2009) Mutations at subsite-3 in cyclodextrin glycosyltransferase from Paenibacillus macerans enhancing alpha-cyclodextrin specificity. Appl Microbiol Biotechnol 83:483–490. doi:10.1007/s00253-009-1865-3 View ArticlePubMedGoogle Scholar
- Li Z, Gu Z, Wang M, Du G, Wu J, Chen J (2010a) Delayed supplementation of glycine enhances extracellular secretion of the recombinant alpha-cyclodextrin glycosyltransferase in Escherichia coli. Appl Microbiol Biotechnol 85:553–561. doi:10.1007/s00253-009-2157-7 View ArticlePubMedGoogle Scholar
- Li ZF, Li B, Gu ZB, Du GC, Wu J, Chen J (2010b) Extracellular expression and biochemical characterization of alpha-cyclodextrin glycosyltransferase from Paenibacillus macerans. Carbohydr Res 345:886–892. doi:10.1016/j.carres.2010.02.002 View ArticlePubMedGoogle Scholar
- Li B, Wang L, Su L, Chen S, Li Z, Chen J, Wu J (2013a) Glycine and Triton X-100 enhanced secretion of recombinant α-CGTase mediated by OmpA signal peptide in Escherichia coli. Biotechnol Bioprocess Eng 17:1128–1134. doi:10.1007/s12257-011-0601-x View ArticleGoogle Scholar
- Li C, Ban X, Gu Z, Li Z (2013b) Calcium ion contribution to thermostability of cyclodextrin glycosyltransferase is closely related to calcium-binding site CaIII. J Agric Food Chem 61:8836–8841. doi:10.1021/jf4024273 View ArticlePubMedGoogle Scholar
- Li Z, Chen S, Gu Z, Chen J, Wu J (2014a) Alpha-cyclodextrin: enzymatic production and food applications. Trends Food Sci Technol 35:151–160. doi:10.1016/j.tifs.2013.11.005 View ArticleGoogle Scholar
- Li Z, Su L, Wang L, Liu Z, Gu Z, Chen J, Wu J (2014b) Novel insight into the secretory expression of recombinant enzymes in Escherichia coli. Prog Biotechnol 49:599–603. doi:10.1016/j.procbio.2014.01.029 Google Scholar
- Liao Y, Lescar J, Tam JP, Liu DX (2004) Expression of SARS-coronavirus envelope protein in Escherichia coli cells alters membrane permeability. Biochem Biophys Res Commun 325:374–380. doi:10.1016/j.bbrc.2004.10.050 View ArticlePubMedGoogle Scholar
- Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60:512PubMedPubMed CentralGoogle Scholar
- Mana RC, Ismailc AF, Ghazalia NF, Fuzi SFZM, Illias RM (2015) Effects of the immobilization of recombinant Escherichia coli on cyclodextrin glucanotransferase (CGTase) excretion and cell viability. Biochem Eng J 98:91–98. doi:10.1016/j.bej.2015.02.013 View ArticleGoogle Scholar
- Mergulhao FJ, Summers DK, Monteiro GA (2005) Recombinant protein secretion in Escherichia coli. Biotechnol Adv 23:177–202. doi:10.1016/j.biotechadv.2004.11.003 View ArticlePubMedGoogle Scholar
- Rosso AM, Ferrarotti SA, Krymkiewicz N, Nudel BC (2002) Optimisation of batch culture conditions for cyclodextrin glucanotransferase production from Bacillus circulans DF 9R. Microb Cell Fact 1:3. doi:10.1186/1475-2859-1-3 View ArticlePubMedPubMed CentralGoogle Scholar
- Roy A, Sc M, Saha S, Sc M, Roy MN (2017) Exploration of inclusion complexes of probenecid with α and β-cyclodextrins: enhancing the utility of the drug. J Mol Struct 1144:103–111. doi:10.1016/j.molstruc.2017.05.002 View ArticleGoogle Scholar
- Sonnendecker C, Wei R, Kurze E, Wang JP, Oeser T, Zimmermann W (2017) Efficient extracellular recombinant production and purification of a Bacillus cyclodextrin glucanotransferase in Escherichia coli. Microb Cell Fact 16:87. doi:10.1186/s12934-017-0701-1 View ArticlePubMedPubMed CentralGoogle Scholar
- Su LQ, Chen S, Yi L, Woodard RW, Chen J, Wu J (2012) Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3). Microb Cell Fact 11:8. doi:10.1186/1475-2859-11-8
- Tonkova A (1998) Bacterial cyclodextrin glucanotransferase. Enzyme Microb Technol 22:678–686. doi:10.1016/s0141-0229(97)00263-9 View ArticleGoogle Scholar
- Yamabhai M, Emrat S, Sukasem S, Pesatcha P, Jaruseranee N, Buranabanyat B (2008) Secretion of recombinant Bacillus hydrolytic enzymes using Escherichia coli expression systems. J Biotechnol 133:50–57. doi:10.1016/j.jbiotec.2007.09.005 View ArticlePubMedGoogle Scholar
- Yang JB, Moyana T, Mackenzie S, Xia Q, Xiang J (1998) One hundred seventy-fold increase in excretion of an FV fragment-tumor necrosis factor alpha fusion protein (sFV/TNF-alpha) from Escherichia coli caused by the synergistic effects of glycine and Triton X-100. Appl Environ Microbiol 64:2869–2874PubMedPubMed CentralGoogle Scholar