Microsoft Word - Jamrichova a kol NBC.doc Nova Biotechnologica et Chimica 14-2 (2015) 201 DOI 10.1515/nbec-2015-0027 © University of SS. Cyril and Methodius in Trnava OPTIMIZATION OF EXPRESSION CONDITIONS OF THE ACETYLESTERASE CE16 FROM Hypocrea jecorina ENCODED BY A SYNTHETIC GENE AND EXPRESSED IN Escherichia coli CELLS DANIELA JAMRICHOVÁ1, ANDREJ GODÁNY1,2, ĽUBICA URBÁNIKOVÁ2 1Department of Biology, University of SS. Cyril and Methodius, J. Herdu 2, Trnava, SK-917 01, Slovak Republic 2Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, Bratislava, SK-845 51, Slovak Republic Abstract: Acetylesterase CE16 was identified as a part of the enzymatic cocktail secreted by fungus Hypocrea jecorina (anamorph: Trichoderma reesei) during its growth on cellulose. Later it was classified as the first member of a newly organized carbohydrate esterase family CE16. Further studies showed that acetylesterase is crucial for complete deacetylation of naturally acetylated xylans enabling their saccharification by xylanases. To study the relationship between structure and function of acetylesterase, highly purified recombinant enzyme produced by Trichoderma reesei Rut C-30 was prepared. The enzyme was composed of 348 amino acid residues from which the 1 - 19 formed a secretion signal peptide. Determined molecular mass of purified recombinant acetylesterase (Aes1) was 45 kDa which was more than molecular mass calculated from amino acid sequence. As it has been proved later, the difference was caused by the enzyme glycosylation. Glycosylation of proteins increases their stability, but it can also be a source of heterogeneity, which might be a problem during crystallization. To make the future X-ray study of the enzyme easier, recombinant non-glycosylated enzyme needed to be prepared. For these purposes, a synthetic gene optimized for protein expression in Escherichia coli was designed and synthetized. The first non- glycosylated acetylesterase obtained by the expression of its synthetic gene in E. coli cells was mostly insoluble or aggregated. Conditions of cell cultivation, induction of gene expression and cells disruption were necessary to optimize. Presently, after optimization of all mentioned steps, the non-glycosylated recombinant CE16 acetylesterase was prepared in the soluble and active form, ready for further downstream procedures, involving protein purification and crystallization. Key words: acetylesterase CE16, Hypocrea jecorina, Trichoderma reesei, synthetic gene, gene expression, Escherichia coli 1. Introduction Filamentous fungus Hypocrea jecorina (anamorph: Trichoderma reesei) is a representative organism today for commercial scale production of different cellulases and hemicellulases, which are currently used for production of biofuels, textile materials, detergents, food, feed and have negligible use in brewing industry. Genus Hypocrea jecorina has become a subject of intense studies from 1980 until now. With the increasing need to replace fossil fuels by cheaper and greener sources of energy, such as constantly forming plant biomass, scientific community has drawn attention to the enzymes produced by this fungus during its growth on cellulose and lactose, to the so called secretome (ADAV et al., 2011). Plant cell wall has a complex structure and Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:23 UTC 202 Jamrichová, D. et al. composition, and therefore its decomposition requires a complex interaction of a range of enzymes cleaving specific types of chemical bonds (CAFFALL and MOHNEN, 2009). Xylan, a constituent of the cell walls, is a polysaccharide made from xylose units which are usually decorated by side substituents, mostly arabinose, D-glucuronic acids or acetyl groups which influences its properties and protects plant cell walls towards hydrolysis by glycoside hydrolases, i.e. acetylation slows down its subsequent digesting (CARPITA and GIBEAU, 1993). The degree of acetylation influences the enzymatic hydrolysis, since esterified polysaccharides are less soluble and less accessible to the enzymatic hydrolysis. One of the enzymes that are able to deacetylate oligosaccharides in the plant cell wall is acetylesterase (TrCE16). Acetylesterase (TrCE16) was identified for the first time in 1985 as a part of the cellulolytic enzyme cocktail produced by H. jecorina during its growth on cellulose (BIELY et al., 1985). According its substrate specificity, the enzyme was classified as an acetic-ester acetyl hydrolase (EC 3.1.1.6) (POUTANEN and SUNDBERG, 1988). Mentioned acetylesterase is regioselective enzyme. Moreover, it is unique by its ability to deacetylate terminally acetylated xylooligosaccharides, predominantly at position 3 and 4, from their non-reducing ends and less effectively at position 2 (KREMNICKÝ and BIELY, 2005; BIELY et al., 2011, BIELY et al., 2014). Further studies showed that acetylesterase efficiently catalyzes transacetylation in aqueous medium saturated with vinyl acetate to the position 3 of non-reducing residues of xylooligosaccharides; and it is a crucial enzyme for complete deacetylation of naturally acetylated xylans enabling their saccharification by xylanases (KREMNICKÝ and MASTIHUBA, 2004; KREMNICKÝ and BIELY, 2005). Because of the need of large quantity of acetylesterase, an acetylesterase gene from wild-type Trichoderma reesei QM 6a was cloned and transformed into hypercellulolytic mutant Trichoderma reesei Rut C-30 as a production strain. Based on amino acid sequence of recombinant acetylesterase (Aes1), a new family of carbohydrate esterases, family CE16 (CAZy classification, LOMBARD et al., 2014), was established in 2008 (LI et al., 2008). Up to date (10 November 2015), the family CE16 comprises 190 members. Unfortunately, there is no tertiary structure information about any member yet. Moreover, only three members are biochemically characterized: acetylesterases from T. reesei (LI et al., 2008), Myceliopthora thermophila (KOUTANIEMI et al., 2013) and from Podospora anserina (PUCHART et al., 2015). Experimentally determined molecular mass of the purified recombinant acetylesterase (Aes1) was 45 kDa (LI et al., 2008), what matched with the molecular mass of purified non- recombinant acetylesterase TrCE16 (POUTANEN and SUNDBERG, 1988), but was lower than molecular mass calculated from amino acid sequence. This difference is caused by the enzyme glycosylation. The fact that natural and recombinant acetylesterases can be N-glycosylated was confirmed by enzymatic cleavage by endo-β-N-acetylglucosaminidase, resulting in the production of lower-mass polypeptide. Glycosylation of proteins increases their stability, but it can be also a source of the heterogeneity, which might cause problems during protein crystallization. Besides this, the transformed strain of T. reesei produces several other acetylesterases similar to our target enzyme, which may cause problems at purification of our target acetylesterase. The problems could be minimized by the production of non-glycosylated recombinant Aes1 protein in Escherichia coli cells. For these purposes, the synthetic Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:23 UTC Nova Biotechnologica et Chimica 14-2 (2015) 203 gene encoding acetylesterase was designed (URBÁNIKOVÁ, unpublished data) and prepared (GenScript, Piscataway, USA). Considering further studies on the relationship between structure and function of acetylesterase, it would seem necessary to prepare a non-glycosylated recombinant enzyme, making easier its crystallization and tertiary structure determination. The advantages of the E. coli expression system are as following: e.g. high overexpression, relatively easy production of proteins and ability to produce proteins with His6-tag sequence, which is helpful in protein purification by IMAC (Immobilized Metal Affinity Column), and, finally, the fact that E. coli does not possess the glycosylation machinery. Heterologous expression is not easy to be performed. High yield and solubility of final protein are influenced by the properties of given enzyme (BASHIRI et al., 2015). In this paper, the expression conditions of the synthetic gene are optimized, with the aim to reach the maximal amount of a soluble active protein. 2. Materials and methods 2.1 Bacterial strains and plasmid Synthetic gene (GenScript, Piscataway, USA) was prepared pursuant to gene aes1 encoding CE16 acetylesterase (Aes1), but without the part coding signal peptide. Codon usage of the synthetic gene was optimized for E. coli cells using the program JCat (GROTE et al., 2005). Optimized synthetic gene tae1 was inserted into pET21a (Novagen, USA) expression vector. The NdeI and XhoI restriction enzymes were used to digest the sequence of the synthetic gene and pET21a vector and the recombinant pET21a-tae1 molecule was prepared by subsequent ligation. The pET21a-tae1 plasmid was transformed by heat-shock method to E. coli competent cells (FROGER and HALL, 2007). Four types of competent cells were used for testing of the gene expression: strain BL21(DE3), NiCo21(DE3), ArcticExpress and BL21-Gold(DE3) (Agilent Technologies, USA). 2.2 Media and culture conditions For cells cultivation, LB-Miller medium (MANIATIS et al., 1982) was used, containing 1 % (w/v) bacto-tryptone (Serva, Germany), 0.5 % (w/v), yeast extract (Difco Laboratories, USA), and 1 % (w/v) NaCl, pH 7.5. In order to enhance the production of soluble recombinant protein LB medium containing 1 M NaCl was prepared (NAGATA et al., 2005, SASAKI et al., 2006, SASAKI et al., 2012). All cultivation media were supplemented with appropriate antibiotics. The transformed cells were grown at 37 °C in 500 ml shake flasks on the shaker at 250 rpm until the OD600 = 0.6-1.0 was reached (about 2.5 h). Then, the temperature in the shaker was lowered to 20° C for 30 minutes and the gene expression was induced by the addition of IPTG (isopropyl–thio–β–D–1–galactopyranoside) to the final concentration of 1 mM (Serva, Germany). Afterwards, several different incubation temperatures and length of cultivation were set and tested: (i) 16 °C for 24 h, (ii) 30 °C for 6 h, and (iii) 11 °C for 24 h when ArcticExpress competent cells were used. As a control of the Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:23 UTC 204 Jamrichová, D. et al. level of protein expression, samples with no IPTG added were prepared at all conditions. In order to improve the production of soluble and well folded recombinant protein, a heat shock was tested, i. e. bacterial culture was exposed to 47 °C for 30 min followed by cooling to 20° C before the IPTG addition (OGANESYAN et al., 2007., BURGESS and MURRAY, 2009). The protein expression was analyzed in the induced cultures and non-induced controls in the adjusted time intervals (6, 20, 24 h during cultivation) by sodium dodecyl sulfate polyacrylamide gel electrophoresis (12 % SDS- PAGE, LAEMMLI, 1970). Proteins in the gel were stained with Coomassie Brilliant Blue (Serva, Germany). After incubation, the cells were harvested by centrifugation at 10,600 g for 15 min at 4 °C and stored at -20 °C for further procedures. 2.3 Cell disruption The cells were disrupted by two methods. At first, (i) chemical lysis by BugBuster Master Mix (5 ml / 1 g of pelleted cells) (Merck Millipore, USA) was performed. However, this type of cell disruption led to formation of soluble aggregates not able to bind to IMAC column. Therefore, (ii) mechanical method of disruption cell walls by sonicator (Soniprep 150, United Kingdom) was used for complete cell lysis. The cells were thawed on ice and then resuspended in a sonication buffer composed of 20 mM sodium phosphate buffer, pH 7.5, supplemented with NaCl and glucose to a final concentration of 200 mM and 25 mM. For the protein solubility improvement various detergents and additives in sonication buffer were tested. As a result, a detergent cocamidopropyl betaine, (Tego Betain F-50, Evonik Goldschmidt GmbH, Germany) and reducing agent TCEP (tris (2-carboxyethyl) phosphine) (Sigma-Aldrich, Germany) were added to the sonication buffer to the final concentration of 1 % and 10 mM, respectively. The resuspended pellet was sonicated using 15 cycles of 30 s pulses at amplitude 14 with 1 min breaks and kept on ice during the whole sonication to avoid protein denaturation. Optimal conditions of sonication were determined by time course analysis of sonication by sampling. The samples for analysis were removed during the sonication and then centrifuged for 5 min at 10,600 g at 4 °C. Protein concentration was determined in supernatant by spectrophotometric analysis at the wavelength of 280 nm (Nanodrop 2000c, Thermo Scientific, USA). After sonication, the lysate was clarified by centrifugation at 10,600 g for 15 min at 4 °C. The samples taken from both, soluble and insoluble fractions, were analyzed by 12 % SDS-PAGE (LAEMMLI, 1970) with SPECTRATM MULTICOLOR BROAD PROTEIN LADDER (10-260 kDa) (SM1841, Thermo Scientific, USA) used as a protein molecular weight control. The proteins bands were visualized by Coomassie Brilliant Blue staining protocol. 3. Results and discussion In this work, a synthetic gene encoding Trichoderma reesei acetylesterase CE16 (tae1) was prepared, according to the sequence of Aes1 (GenBank DQ866149), without a part encoding the signal peptide (Fig. 2). Codon usage was optimized for the Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:23 UTC Nova Biotechnologica et Chimica 14-2 (2015) 205 expression in E. coli cells in order to overcome problems originating from the differences between the eukaryotic gene and the prokaryotic host organism (JONASSON et al, 2002). The prepared gene was inserted into the pET21a vector with C-terminal His6-tag sequence and a gene responsible for ampicillin resistance (Fig. 1). In general, this vector enables the expression under the control of a strong T7 promotor and also provides multi cloning site (MCS) with restriction enzymes NdeI and XhoI used for the insertion of the synthetic gene tae1 (Fig. 1). Fig. 1. The construct of a pET21a expression vector with inserted synthetic gene tae1. Inserted synthetic gene is marked by red color. Nucleotide sequence of the synthetic gene tae1 with modified nucleotides in red is placed in the red box. Tae1 ------------------MFPKPHDDFKYLITFGDSYTDNGRLGYYGSHQAHGPPPGVMP Aes1 MRSILVIPSFVAVLNAFSLFPKPHDDFKYLITFGDSYTDNGRLGYYGSHQAHGPPPGVMP :***************************************** Tae1 PEANVTASGGLQWPQYVEASTGATLYDYAIAGATCDNNNVERWAAFMNANYPSIITDEIP Aes1 PEANVTASGGLQWPQYVEASTGATLYDYAIAGATCDNNNVERWAAFMNANYPSIITDEIP ************************************************************ Tae1 SFKADRKTKLYRGVTSANTVYALWIGTNDLSYTGILSDSQVKGTNITTYIDCLWNVFDAI Aes1 SFKADRKTKLYRGVTSANTVYALWIGTNDLSYTGILSDSQVKGTNITTYIDCLWNVFDAI ************************************************************ Tae1 HAAGGRRFVILNNNALQLTGLYRPLSDGGAGDNQFWQNKTLYNQTEYAQKMLEYTTSSNT Aes1 HAAGGRRFVILNNNALQLTGLYRPLSDGGAGDNQFWQNKTLYNQTEYAQKMLEYTTSSNT ************************************************************ Tae1 MIDYGVPFHLLVKNRWPGSKVAVYDIHSLIMDIYNQPSRYLEPPHNVVGYYKHCDVNGTN Aes1 MIDYGVPFHLLVKNRWPGSKVAVYDIHSLIMDIYNQPSRYLEPPHNVVGYYKHCDVNGTN ************************************************************ Tae1 CLYGPGRLDSYLWYDELHPSNIIASYIAREFLNVVSGRSKYGTYWEHWLEHHHHHH Aes1 CLYGPGRLDSYLWYDELHPSNIIASYIAREFLNVVSGRSKYGTYWEHW-------- ************************************************ Fig. 2. Alignment of amino acid sequences of the two recombinant acetylesterases CE16. Tae1 = non- glycosylated acetylesterase produced by E.coli, Aes1 = glycosylated acetylesterase produced by Trichoderma reesei RutC-30, for the alignment Clustal Omega online tool was used (SIEVERS and HIGGINS, 2011). In the Tae1 sequence, amino acid residues added due to the cloning strategy are in red and His6-tag is in green color. In Aes1 sequence, amino acid residues corresponding to the signal peptide are in blue. N-glycosylation sites are in light blue color in both sequences. accgtttacgctctgtggatcggtaccaacgacctgtcttacaccggtatcctgtctgactctcaggttaaaggtaccaacatcaccacc tacatcgactgcctgtggaacgttttcgacgctatccacgctgctggtggtcgtcgtttcgttatcctgaacaacaacgctctgcagctg accggtctgtaccgtccgctgtctgacggtggtgctggtgacaaccagttctggcagaacaaaaccctgtacaaccagaccgaatacgct cagaaaatgctggaatacaccacctcttctaacaccatgatcgactacggtgttccgttccacctgctggttaaaaaccgttggccgggt tctaaagttgctgtttacgacatccactctctgatcatggacatctacaaccagccgtctcgttacctggaaccgccgcacaacgttgtt ggttactacaaacactgcgacgttaacggtaccaactgcctgtacggtccgggtcgtctggactcttacctgtggtacgacgaactgcac ccgtctaacatcatcgcttcttacatcgctcgtgaattcctgaacgttgtttctggtcgttctaaatacggtacctactgggaacactgg atgttcccgaaaccgcacgacgacttcaaatacctgatcaccttcggtgactcttacaccgacaacggtcgtctgggttactacggttct caccaggctcacggtccgccgccgggtgttatgccgccggaagctaacgttaccgcttctggtggtctgcagtggccgcagtacgttgaa gcttctaccggtgctaccctgtacgactacgctatcgctggtgctacctgcgacaacaacaacgttgaacgttgggctgctttcatgaac gctaactacccgtctatcatcaccgacgaaatcccgtctttcaaagctgaccgtaaaaccaaactgtaccgtggtgttacctctgctaac Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:23 UTC 206 Jamrichová, D. et al. Fig. 3. Analysis of protein production by different E.coli strains. SDS - PAGE with samples taken at different time of the protein production in selected E. coli cells: A - BL21(DE3)-pET21a-tae1; B - NiCo21(DE3)-pET21a-tae1, C - BL21-Gold-(DE3)-pET21a-tae1; D - ArcticExpress cells-pET21a-tae1. Ladder: SPECTRATM MULTICOLOR BROAD PROTEIN LADDER (10-260 kDa) (Thermo Scientific, USA). Control = cultivation without induction. In the first experiments with a production of the non-glycosylated acetylesterase obtained by the expression of its synthetic gene in E. coli cells, the problems with protein solubility were found. Production of deserved protein was satisfying, but after cell homogenization, the protein was mostly insoluble or aggregated and not able to bind to IMAC column. These problems suggested incorrect folding of the protein 1. 2. 3. 4. 5. 6. 7. 8. 1. 2. 3. 4. 5. 6. 7. 8. 40 kDa 35 kDa Tae1 38.29 kDa 40 kDa 35 kDa 1. 2. 3. 4. 5. 6. 7. 8. Tae1 38.29 kDa Legend: 1. cells before induction 2. protein ladder 3. 6 h control 4. 6 h- IPTG induction 5. 20 h control 6. 20 h- IPTG induction 7. 24 h control 8. 24 h- IPTG induction Legend: 1. cells before induction 2. 6 h control 3. 6 h- IPTG induction 4. 20 h control 5. 20 h- IPTG induction 6. 24 h control 7. 24 h- IPTG induction 8. protein ladder 40 kDa Tae1 38.29 kDa D A Legend: 1. cells before induction 2. 6 h control 3. 6 h- IPTG induction 4. 20 h control 5. 20 h- IPTG induction 6. 24 h control 7. 24 h- IPTG induction 8. protein ladder Legend: 1. protein ladder 2. cells before induction 3. 6 h control 4. 6 h- IPTG induction 5. 20 h control 6. 20 h- IPTG induction 7. 24 h control 8. 24 h- IPTG induction 35 kDa 1. 2. 3. 4. 5. 6. 7. 8. B 1. 2. 3. 4. 5. 6. 7. 8. 35 kD 40 kDa Tae1 38.29 kDa B Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:23 UTC Nova Biotechnologica et Chimica 14-2 (2015) 207 (HANDRICK and HARTL, 1995). With the aim to improve the recombinant protein folding and solubility, at first, different E. coli expression strains were tested, and the choice of given strains for the gene expression was closely tied to the properties of the selected expression vector. Different types of expression cells were used to study the gene expression level. BL21(DE3) and NiCo21(DE3) were used as standard types of competent cells. ArcticExpress competent cells were used due to their adjustment of low-temperature cultivation in order to increase the recovery of the soluble protein (SCHEIN, 1989). BL21-Gold(DE3) competent cells are designed for easy induction and high-level protein expression. The pET21a-tae1 plasmid was transformed into E. coli cells followed by the cultivation in a standard manner by described method. Gene expression was monitored by SDS-PAGE analysis. The obtained results did not show any significant differences in the synthetic gene expression among the tested BL21(DE3) (Fig.3; A), NiCo21(DE3) (Fig.3, B) and BL21-Gold(DE3) (Fig.3; C) expression cells. To emphasize, in each step of protein production and protein extraction from the cells, the total protein content was indirectly confirmed spectrophotometrically by measuring the absorbance at 280 nm. The cells were disrupted by BugBuster, but the recombinant protein was found either in the insoluble part or formed soluble aggregates and was not able to bind to IMAC column. For this reason, mechanical disruption by sonication was tested, but the majority of protein also stood in the insoluble part. The total amount of protein produced by ArcticExpress competent E.coli cells (Fig.3, D) was lower than in other three cases; and after homogenization, the protein remained mostly in the insoluble fraction. Therefore, these competent cells were not used in further procedures. Fig. 4. SDS - PAGE with samples taken in different time of the protein production in BL21-Gold-(DE3) in LB medium supplemented by 1 M NaCl and with a heat-shock before induction. Ladder: SPECTRATM MULTICOLOR BROAD PROTEIN LADDER (10-260 kDa) (Thermo Scientific, USA). Control = cultivation without induction. In order to improve protein solubility and reach a high level of protein production, conditions of cultivation and cell lysis were optimized. Many recombinant proteins cannot be expressed well and in a correct and soluble form in E. coli cells because of the absence of the proper folding environment or the absence of the post translational modification (SORENSEN et al; 2005). Presently, various defense mechanisms of bacteria are known to protect proteins from aggregation and incorrect folding by 1. 2. 3. 4. 5. 6. 7. 8. 40 kDa 35 kDa Tae1 38.29 kDa Legend: 1. cells before induction 2. 6 h-control 3. 6 h- IPTG induction 4. 20 h control 5. 20 h- IPTG induction 6. 24 h control 7. 24 h- IPTG induction 8. protein ladder Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:23 UTC 208 Jamrichová, D. et al. osmotic or heat stress. Defense mechanisms include intracellular accumulation of osmolytes or synthesis of heat shock proteins in the cells (KEMPF et al; 1998; BUKAU et al; 1998). Therefore, several cultivation conditions using the high concentration of NaCl (1 M) in LB medium and a heat shock (47 °C, 30 min) before induction were applied. In the view of increased temperature during heat shock and the length of cultivation, ampicillin was replaced by its stable equivalent carbenicillin. From the protein solubility point of view, the most soluble protein was obtained after 20 h cultivation at 16 °C in salted LB medium with heat shock prior to induction (Fig. 4). The conditions for improved protein solubility were adjusted by adding Tego Betain F50 and TCEP, both before sonication. TCEP is a reducing agent that selectively reduces disulphide bonds and that is nonreactive toward other functional groups commonly present in proteins (KIRLEY et al; 1989). Moreover TCEP is compatible with IMAC column containing Ni2+ ions. Tego Betain F50 is a zwitterionic detergent containing a quaternary ammonium cation and a carboxylate groups, shown aid in protein stability and folding (NITSCH et al; 2005). The disadvantage of Tego Betain is high foaming, which may cause protein denaturation. To avoid denaturation and obtain the highest possible yield of the soluble protein, the concentration of Tego Betain and TCEP in sonication buffer, as well as a volume of sonication buffer per gram of cells were optimized. The number of sonication cycles (Fig. 5) was also optimized in all sonicated samples to avoid the protein denaturation; the temperature during the whole period of sonication was controlled to prevent well-known damages caused by ultrasound radiation in biological system (WOOD and LOOMIS, 1927). The best result of both, well-expressed and most soluble protein was achieved by protein production using E. coli BL21-Gold(DE3) cells as a host, mechanical cell disruption by sonication using the buffer supplemented with Tego Betain F-50 and TCEP to the final concentration of 1 % and 10 mM, respectively (Fig. 6) and subjecting the cells to 15 cycles of sonication (15 x 15 s) with one minute breaks for better cooling of the sample. Above-mentioned conditions were evaluated as the best conditions for acetylesterase CE16 production in terms of the protein yield and solubility. Fig. 5. Determination of optimal conditions of cell disruption by sonication. Samples were collected after each three cycles of sonication. Ladder: SPECTRATM MULTICOLOR BROAD PROTEIN LADDER (10- 260 kDa) (Thermo Scientific, USA). Legend: 1. TCP (Total cell fraction) 2. 3 x 15 s 3. 6 x 15 s 4. 9 x 15 s 5. 12 x 15 s 6. 15 x 15 s 7. 18 x 15 s 8. 19 x 15 s 9. protein ladder 1. 2. 3. 4. 5. 6. 7. 8. 9. 40 kDa 35 kDa Tae1 38.29 kDa Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:23 UTC Nova Biotechnologica et Chimica 14-2 (2015) 209 Fig. 6. Influence of sonication buffer composition on the yield of soluble protein. The comparison of the sonication results analyzed on SDS-PAGE. A - sonication buffer without TCEP and Tego Betain; B - sonication buffer containing TCEP and Tego Betain. Ladder: SPECTRATM MULTICOLOR BROAD PROTEIN LADDER (10-260 kDa) (Thermo Scientific, USA). 4. Conclusions In the present work, conditions of recombinant molecule pET21-tae1 expression and soluble recombinant acetylesterase production, i.e. conditions of cell cultivation, induction of protein expression and sonication of cell pellet were optimized. E. coli BL21GOLD(DE3) strain was chosen for its property to maximally express the synthetic gene encoding acetylesterase Tae1 and produce the protein in soluble form. The cultivation of E. coli cells BL21GOLD(DE3) containing recombinant pET21a-tae1 construct proceeded after heat shock, until exponential phase was reached. Then the expression of the gene encoding CE16 acetylesterase was inducted by adding IPTG to the final concentration of 1 mM, in LB medium containing 1 M NaCl for 20 h at 16 °C. Collected E. coli cells (after storing at -20 °C) were sonicated; and solubilized protein was obtained by sonication in buffer supplemented with TCEP reducing agent and Tego Betain F50, zwitterionic detergent. The best results were achieved after 15 cycles of 30 s pulses at amplitude 14. At this stage, a non- glycosylated recombinant CE16 acetylesterase was prepared as a soluble protein fraction after sonication under specific conditions, and is stored for further downstream procedures, involving especially protein isolation by Immobilized Metal Affinity Column (IMAC), purification by size exclusion chromatography using FPLC system and crystallization for the tertiary structure determination. Acknowledgement: The work was supported by the Slovak Research and Development Agency grant APVV-0602-12, by the Slovak Grant Agency VEGA grant No. 2/0190/14 and by the Faculty of Natural Sciences of UCM under the contract no. FPPV-27-2015. The authors thank to Lenka Tišáková for the language checking. References ADAV, S.S., RAVINDRAN, A., CHAO, L.T., TAN, L., SINGH, S., SZE, S.K.: Proteomic analysis of pH and strains dependent protein secretion of Trichoderma reesei. J. Proteome Res., 10, 2011, 4579 - 4596. A Tae1 38.29 kDa 40 kDa 35 kDa Legend: 1. TCP (Total cell fraction) 2. soluble fraction 3. insoluble fraction 4. protein ladder 1. 2. 3. 4. Tae1 38.29 kDa 35 kDa 40 kDa Legend: 1. TCP (Total cell fraction) 2. soluble fraction 3. insoluble fraction 4. protein ladder 1. 2. 3. 4. B B A B Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:23 UTC 210 Jamrichová, D. et al. BASHIRI, S., VIKSTROM, D., ISMAIL, N.: Optimization of protein expression in Escherichia Coli. Biopharm. J., 28, 2015, 42 - 44. BIELY, P., CZISZÁROVÁ, M., AGGER, J.W., LI, X.L., PUCHART, V., VRŠANSKÁ, M., EIJSINK, V.G., WESTERENG, B.: Trichoderma reesei CE16 acetyl esterase and its role in enzymatic degradation of acetylated hemicellulose. Biochim. Biophys. Acta, 1840, 2014, 516 - 525. BIELY, P., MASTIHUBOVÁ, M., TENKANEN, M., EYZAGUIRRE, J., LI, X.L., VRŠANSKÁ, M.: Action of xylan deacetylating enzymes on monoacetyl derivatives of 4-nitrophenyl glycosides of β-D-xylopyranose and α-L- arabinofuranose. J. Biotechnol., 151, 2011, 137 - 142. BIELY, P., PULS, J., SCHNEIDER, H.: Acetyl xylan esterases in fungal cellulolytic systems. FEBS Letters, 186, 1985, 80 - 84. BUKAU, B., HORWICH, A.L.: The Hsp70 and Hsp60 chaperone machines. Cell J., 92, 1998, 351 - 366. BURGESS, R.R., MURRAY, P.D.: Guide to protein purification. Elsevier Inc., San Diego, 2009, 851 pp. CAFFALL, K.H., MOHNEN, D.: The structure, function and biosynthesis of plant cell wall pectic polysaccharides. Carbohydr. Res., 344, 2009, 1879 - 1900. CARPITA, N.C., GIBEAUT, D.M.: Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J., 3, 1993, 1 - 30. FROGER, A., HALL, J.E.: Transformation of Plasmid DNA into E. coli Using the Heat Shock Method. J. Vis. Exp., 6, 2007, http://www.jove.com/index/ Details.stp?ID=253, DOI: 10.3791/253. GROTE, A., HILLER, K., SCHEER, M., MUNCH, R., NORTEMANN, B., HEMPEL, D.C., JAHN, D.: JCat: a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res., 1, 2005, 526 -5 31. HANDRICK, J.P., HARTL, F.U.: The role of molecular chaperones in protein folding. FASEB J., 9, 1995, 1559 - 1569. JONASSON, P., LILJEQVIST, S., NYGREN, P., STAHL, S.: Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli. Biotechnol. Appl. Biochem., 35, 2002, 91–105. KEMPF, B., BREMER, E.: Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments. Arch. Microbiol., 170, 1998, 319 - 330. KIRLEY, T.L.: Reduction and fluorescent labeling of cyst(e)ine-containing proteins for subsequent structural analysis. Anal. Biochem., 180, 1989, 231 - 236. KOUTANIEMI, S., VAN-GOOL, M. P., JUVONENA, M., JOKELAC, J., HINZ, S. W., SCHOLS, H. A., TENKANENA, M.: Distinct roles of carbohydrate esterase family CE16 acetylesterases and polymer-acting acetyl xylan esterases in xylan deacetylation. J. Biotechnol., 168, 2013, 684 - 692. KREMNICKÝ, L., BIELY, P.: Unique mode of acetylation of oligosaccharides in aqueous two-phase system by Trichoderma reesei acetylesterase. J. Mol. Catal. B: Enzym., 37, 2005, 72 - 78. KREMNICKÝ, L., MASTIHUBA, V., COTE, G.L.: Trichoderma reesei acetylesterase catalyzes transesterification in water. J. Mol. Catal. B: Enzym., 30, 2004, 229 - 239. Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:23 UTC Nova Biotechnologica et Chimica 14-2 (2015) 211 LAEMMLI, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 1970, 680 - 685. LI, X.L., SKORY, C.D., COTTA, M.A., PUCHART, V., BIELY, P.: Novel family of carbohydrate esterases, based on identification of the Hypocrea jecorina acetylesterase gene. Appl. Environ. Microbiol., 74, 2008, 7482 - 7489. LOMBARD, V., GOLACONDA, R.H., DRULA, E., COUTINHO, P.M, HENRISSAT, B.: The Carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res., 42, 2014, 490 - 495. MANIATIS, T., FRITSCH, E.F., SAMBROOK, J.: Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory, 1982, 545 pp. NAGATA, S., SASAKI, H., OSHIMA, A., TAKEDA, S., HASHIMOTO, Y., ISHIDA, A.: Effect of proline and K+ on the stimulation of cellular activities in Escherichia coli K-12 under high salinity. Biosci. Biotechnol. Biochem., 69, 2005, 740 - 746. NITSCH, CH., HEITLAND, H.J., MARSEN, H., SCHLÜUSSLER, H.J.: Ullmann’s Encyclopedia of Industrial Chemistry, Wiley-VCH, Düsseldorf, 2003, 3008 pp. OGANESYAN, N., ANKOUDINOVA, I., KIM, S.H., KIM R.: Effect of Osmotic Stress and Heat Shock in Recombinant Protein Overexpression and Crystallization. Protein Expr. Purif., 52, 2007, 280 - 285. POUTANEN, K., SUNDBERG, M.: An acetylesterase of Trichoderma reesei and its role in the hydrolysis of acetyl xylans. Appl. Microbiol. Biotechnol., 28, 1988, 419 - 424. PUCHART, V., BERRIN, J.G., HAON, M., BIELY, P.: A unique acetylesterase from Podospora anserina active on polymeric xylan. Appl. Microbiol. Biotechnol., 2015, 1 - 12. SASAKI, H., ISHIDA, A., HASHIMOTO, Y., TAKEDA, S., OSHIMA, A., KAWAI, H., NAGATA, S.: Utilization of proline in Escherichia coli K-12 at different osmolarities. J. Biol. Sci., 6, 2006, 675 - 679. SASAKI, H., KURIKI, K., OSHIMA, A., ISHIDA, A., NAGATA, S.: Effect of overexpression of proline transporter ProP on high saline adaptation in Escherichia coli. Bull. Soc. Sea Water Sci. Jpn., 66, 2012, 30 - 35. SIEVERS, F., WILM, A., DINEEN, D., GIBSON, T.J., KARPLUS, K., LI, W., LOPEZ, R., MCWILLIAM, H., REMMERT, M., SÖDING, J., THOMPSON, J.D., HIGGINS, D.G.: Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol., 7, 2011, 539. SORENSEN, H.P., MORTENSEN, K.K.: Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb. Cell Fact., 4, 2005, 1 - 8. WOOD, R.W., LOOMIS, A.L.: The physical and biological effects of high frequency sound waves of great intensity. Phil. Mag., 74, 1927, 414 - 436. Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 17.01.20 10:23 UTC