Soluble Expression of the Fucosyltransferase Gene from Helicobacter pylori in Escherichia coli by Co-expression of Molecular Chaperones

샤페론단백질동시발현기술을이용하여 Helicobacter pylori 유래의 fucosyltransferase의수용성생산

Abstract

Fucosyltransferases (FucTs) catalyze fucosyl transfer from guanosine-diphosphate fucose (GDP-β-L-fucose) to acceptor molecules to form fucosyloligosaccharides with α-glycosidic linkages. However, when FucT genes have been expressed in Escherichia coli, most cases have resulted in the production of inclusion bodies. In this study, to overcome this drawback, molecular chaperones were co-expressed with α1,2-fucosyltransferase (FucT2) in E. coli. For this, the pACYC184 vector, having genes for chaperones such as GroEL, GroES, DnaK, DnaJ, and GrpE, were transformed into E. coli BL21 (DE3) star harboring pHFucT2, including the FucT2 gene from Helicobacter pylori 26695. The results from SDS-PAGE showed that 5 chaperones were successfully expressed and the soluble fraction of FucT2 was also increased. HPLC analysis revealed that the coexpression of chaperone proteins resulted in a 5-fold increase in the total activity of fucosyltransferase in E. coli. In conclusion, the FucT2 expression system developed in this study can be used as a useful tool for the synthesis of fucosyloligosaccharides.

Fucosyltransferase는 퓨코실화된 올리고당을 생성하는데 필수적인 효소로서, GDP-β-L-fucose로 부터 fucose를 수용체로 전이시켜 알파 글리코사이드 결합을 형성하는 과정을 촉매한다. 하지만 Escherichia coli 에서 발현시켰을 때, 대부분의 경우 inclusion body를 형성하여 비활성으로 생성되었다. 따라서 본 연구에서는 Helicobacter pylori 26695에서 유래한 α1,2-fucosyltransferase (FucT2) 유전자의 수용성 발현을 위하여, E. coli에서 샤페론 단백질인 GroEL, GroES, DnaK, DnaJ, GrpE과 함께 동시발현시켰다. SDS-PAGE 분석결과, 5가지 샤페론 단백질과 함께 발현되었으며, 수용성 FucT2 단백질이 증가하였고 효소활성은 5배 증가하였다. 결론적으로, 샤페론 동시발현기술을 이용하여 대장균에서 FucT2 수용성 생산을 증가시킬 수 있었으며 본 수용성 효소는 퓨코실화된 올리고당을 효율적으로 생산하는데 이용될 수 있다.

Introduction

Fucosylation, which is the enzymatic transfer of L-fucose from donor molecule to either an oligosaccharide or a protein, is carried out by a class of enzymes called fucosyltransferases (FucTs). FucTs are important class of enzymes for mammals as well as bacteria. In mammalian systems, fucose containing glycoconjugates is directly involved in many biological processes, such as immune responses, cell adhesion, and in many human disease [4, 20, 23]. The reaction also occurs during the synthesis of the ABO(H) and Lewis antigens in human physiology [21, 22] and is commonly observed in the O-antigens present in prokaryotes, the exposed portion of the lipopolysaccharides [13]. Functions arising due to the O-antigens include but are not limited to virulence, molecular mimicry, clearance from the host’s immune system, cell adhesion, and localization [14, 17]. For example, one of the mechanisms by which Helicobacter pylori may escape elimination by the host immune system is the production of surface antigens mimicking those in the host [2].

Based on the new position of glycosidic linkage, FucTs enzymes also are classified into 4 different sub-families (typically α1,2-, α1,3/4-, α1,6- and O-fucTs) [14]. Among them, α1,2-FucTs belong to glycosyltransferase family 11 and are responsible for the transfer of fucose to galactose forming an α1,2-linkage [27]. In previously report, some FucT2 enzymes have been expressed at a soluble secretory form of FT3 (SFT3) in stably-transfected baby hamster kidney cells [7], in insect Spodoptera frugiperda (Sf9) cells using the baculovirus expression system [18], and in transgenic pigs using the human α1,2-fucosyltransferase [6]. When expressed in yeast, Saccharomyces cerevisae, rgalT I has been shown to be N-hyperglycosylated [15]. In fact, S. cerevisiae is known to synthesize polymannans which can considerably impair biological activity of the recombinant proteins. For industrial production of recombinant FucTs, these host systems are not yet compatible with bacterial systems in terms of growth rate, productivity, and cost [5].

E. coli is one of the most extensively used host for the production of homo- and heterologous proteins as it produces large scales of recombinant proteins in rapid, often inexpensive and high-density cultivation [3]. And they also have an advantage of growing quickly in simple media with well characterized and well-known genetics [8, 11, 24]. However, major limitation of this system is that high level expression of recombinant protein often results in aggregation and accumulation in inclusion body. Inclusion body formation is a major drawback for the structural and/or biochemical studies that requires proper folding [16].

In this study, for the active and soluble expression of FucT gene, we attempted to use various molecular chaperones that can assist the folding of newly synthesized proteins to the native state which is helpful to refold misfolded and aggregated proteins [9]. It is widely recognized that the coexpression of molecular chaperones can assist with proper folding of protein, which leads to an increased yield of active protein. In previous studies, the soluble expression of cold-active lipase increased when coexpressed with chaperones [8]. Also, Coexpression of the chaperone team at appropriate levels resulted in marked stabilization and accumulation of Cryj2 [19]. We performed heterologous expression of FucT2 gene from H. pylori 26695 with chaperone genes for GroEL, GroES, DnaK, DnaJ, and GrpE in E. coli.

 

Materials and Methods

Bacterial strains, plasmids, culture conditions, and reagents

E. coli BL21 (DE3) star (Invitrogen, Carlsbad, CA, USA) was used as a host for protein expression, and it was grown aerobically at 37℃ in Luria-Bertani (LB) medium. E. coli transformants were grown in LB medium containing kanamycin (10 μg/ml) at 37℃ for an appropriate time period. The plasmid pHFucT2 containing the FucT2 gene of H. pylori 26695 was previously constructed using plasmid pCOLADuet−1 (Merck Biosciences, Darmstadt, Germany) [12]. The vectors contained kanamycin resistance gene and AraB promoter. The plasmid pKJE7 (7.2 kb) contained DnaK-DnaJ-GrpE genes and the plasmid pG-KJE3 (11.1 kb) had GroEL-ES and DnaK-DnaJ-GrpE genes. The bacterial strains and recombinant DNAs used in this study are listed in Table 1. AccuPrep plasmid mini extraction kit and a molecular weight standard of DNA were purchased from Bioneer Co. (Daejeon, Korea). Protein molecular weight markers were purchased from Bio-Rad (Berkeley, CA, USA). Other chemicals were of reagent grade.

Table 1.List of strains and plasmids used in this study.

Co-expression of fucosyltransferase gene with molecular chaperones

To co-express FucT2 gene with molecular chaperones, 3 kinds of vectors were transformed in E. coli. Each plasmids pair (pKJE7 + pHFucT2 or pG-KJE3 + pHFucT2) was cotransformed in E. coli BL21 (DE3) star competent cell, and the 2 different transformants were obtained on the LB agar plate containing kanamycin and chloramphenicol as selectable marker. They were designated as E. coli (pHFucT2 + pKJE7) and E. coli (pHFucT2 + pG-KJE3). They were cultured at 37℃ in LB medium containing kanamycin (10 μg/ml) and/or chloramphenicol (34 μg/ml). The cultured cell fraction (1 ml) was transferred into 100 ml of fresh LB medium and cultured with shaking until OD600 reached 0.5. Expression of the recombinant gene was induced by 0.03 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (for FucT2) and 0.3 mM L-arabinose (for chaperones). Then the cells were further grown for an additional 12 h at 20℃ with agitating.

Enzyme preparation

After 12 h of induction, cells were collected by centrifugation and the pellet was resuspended in a buffer of 50 mM sodium phosphate buffer (pH 7.0). They were disrupted by ultrasonication (on 35 s, off 15 s) (total fraction) and then cell lysate was obtained by centrifugation at 11,000 × g for 10 min (soluble fraction) and debris (insoluble fraction) was separated for the analysis of inclusion bodies. Insoluble fraction was concentrated into 10-fold (v/v). The expression level of recombinant enzymes was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).

Fucosyltranferase activity assay

Enzyme activity of FucT2 in the crude extract was determined by a thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) by measuring 2'-fucosyllactose. The assay solution consisted of 5 mM GDP-fucose, 25 mM lactose in 50 mM Tris-HCl (pH 5.0), containing 5 mM MnCl2 in a total volume of 1.0 ml. The reaction solution was incubated at 37℃ for 2 h after the addition of 200 μl crude extract solution. The reaction was stopped by boiling for 5 min. For TLC analysis, each of the end products and standards was applied on the plate where 15 mm apart from the bottom (Merck, Darmstadt, Germany). The plate was developed once to the top using 1-propanol/water/acetic acid [2:1:1 (v/v/v)]. After development, the sugars were visualized by dipping the TLC plate into 200 ml of acetone solution containing diphenylamineaniline-phosphoric acid (4 g of diphenylamine, 4 ml of aniline and 20 ml of 85% phosphoric acid) [1]. After air drying, the TLC plate was placed into an oven at 120℃ for 10 min. For HPLC analysis of 2'-fucosyllactose and lactose in culture supernatants, the normal phase HPLC system was used, consisting of a YOUNG-LIN M930 solvent delivery pump (Younglin, Seoul, Korea) with Aminex HPX87-H column (Bio-Rad, Quarry Bay, Hong Kong). The mobile phase was 0.01N H2SO4 in H2O and the flow rate was 0.4 ml/min at 50℃. The analytes were monitored with refractive index (RI) detector (Younglin) [12]. Protein concentration was measured using a Thermo BCATM protein assay kit (Pierce Biotechnology Inc., Rockford, IL, USA).

 

Results and Discussion

Co-expression of pHFucT2 with molecular chaperones

To assess the effect of co-expression of various molecular chaperones on the production of FucT2, SDS-PAGE analysis was performed (Fig. 1). In case of E. coli (pHFucT2) which has no chaperone genes, the gel showed a band corresponding to the FucT2 protein in both total and insoluble fractions, while no band in the soluble fraction of cell lysates, revealing insoluble expression of FucT2. When FucT2 and molecular chaperones were co-expressed in either E. coli (pHFucT2 + pKJE7) or E. coli (pHFucT2 + pG-KJE3), 5 chaperone proteins were successfully expressed and bands for FucT2 were shown in all total, soluble, and insoluble fractions. This result revealed that soluble fraction of FucT2 was increased after co-expression of chaperone genes in E. coli. The soluble expression levels of E. coli (pHFucT2 + pG-KJE3) was higher than that of E. coli (pHFucT2 + pKJE7) and, therefore, we used the former transformant for the next experiments (data not shown).

Fig. 1.SDS-PAGE analysis of co-expressed pHFucT2 and molecular chaperones. E. coli BL21 (DE3) star cells harboring (pHFucT2), (pHFucT2 + pKJE7), and (pHfucT2 + pG-KJE3) were induced with 0.03 mM IPTG for fucosyltransferase (FucT2) gene and 0.3 mM L-arabinose for chaperone expression and grown at 25℃ for 12 h. M, protein marker; T, total fraction; S, soluble fraction; I, insoluble fraction; I10X, 10X insoluble fraction.

Effect of cultivation temperature and period

To increase the soluble and active fractions of the protein during expression, the expression temperature was lowered to 20℃ and 15℃ in order to decrease the protein synthesis rate. Recombinant E. coli (pHFucT2 + pG-KJE3) was pre-cultured up to OD600 0.5 at 37℃ and expression of heterologous proteins were induced with 0.03 mM IPTG and 0.3 mM L-arabinose, respectively, followed by further cultivation for 40 h at 20℃ or 15℃.

As shown in Fig. 2A, when expression temperature was lowered to 15℃, the expression levels of FucT2 in total protein fractions were also decreased, however the thickness level of soluble FucT2 band was increased than that of 20℃ on the gel. This result showed that low temperature was favorable condition for soluble expression proteins. When molecular chaperones were co-expressed at 15℃ and 20℃, soluble fractions bands of FucT2 were thicker than that of the control. This result indicates that soluble FucT2 was more efficiently expressed when it was coexpressed with pG-KJE3 at 15℃ than 20℃. SDS-PAGE results of cultivation period for 20 h (Fig. 2A) and for 40 h (Fig. 2B) showed no significant difference in the soluble expression level of FucT2.

Fig. 2.Effects of cultivation temperature and period on FucT2 expression. Recombinant E. coli harboring (pHFucT2) and (pHfucT2 + pG-KJE3) were grown up to OD600 0.5 at 37℃ and expression of FucT2 and chaperones were induced with 0.03 mM IPTG and 0.3 mM L-arabinose, respectively, followed by further cultivation for (A) 20 h and (B) 40 h at 15℃ and 20℃.

Our experiments showed that lowering the culture temperature to 20℃ or 15℃ increased the soluble expression level of FucT2. The aggregation reaction is in general favored at higher temperatures due to dependence of hydrophobic interactions that determine the aggregation reaction [11]. Sudden decrease in cultivation temperature inhibits replication, transcription, and translation [25] and it lowers the cellular protein concentration which favors folding. However, low temperature also resulted in a slow bacterial growth giving a decreased amount of biomass [26].

Synthesis of 2'-fucosyllactose

After FucT2 protein was co-expressed as soluble form with chaperones in E. coli, detection of the enzyme activity was attempted by using the crude cell extract. Soluble fraction of FucT2 protein was mixed with 25 mM lactose, 5 mM GDP-L-fucose, and 5 mM MnCl2 in 20 mM Tris-HCl buffer at pH 5.0 and incubated 37℃ for 2 h. As shown in Fig. 3, the trisaccharide, 2'-fucosyllactose, was clearly visible to be produced after 2 h of incubation of enzyme and substrates. As shown in Fig. 4, further verification of enzymatic activity was demonstrated by using HPLC. When GDP-L-fucose, lactose, and 2'-fucosyllactose were analyzed by HPLC, they were detected at 16.1, 11.5, and 10.6 min, respectively. The newly synthesized peak with retention time of 10.6 min corresponded to 2'-fucosyllactose, whereas no product peak was observed with the negative control. This result revealed that the reaction products of recombinant FucT2 is 2'-fucosyllactose.

Fig. 3.TLC analysis result of 2-FL production by using crude fucosyltransferase enzymes. 1: GDP-L-fucose; 2: lactose; 3: 2'-fucosyllactose; 4, Standard Mixture; 5, control, no enzyme; 6, 7, reaction products after adding crude extract derived from E. coli (pHfucT2) cells; 8, 9, reaction products after adding crude extract derived from E. coli (pHfucT2 + pG-KJE3) cells.

Fig. 4.HPLC chromatograms of 2-FL production by using crude fucosyltransferase enzyme. (A) standard materials; 1,2'-fucosyllactose; 2, lactose; 3, GDP-L-fucose (B) control, no enzyme; (C) reaction products after adding crude extract derived from E. coli (pHfucT2 + pG-KJE3) cells.

Enzyme activity of fucosyltransferase

The fucosyltransferase activity in the crude cell extract of recombinant E. coli (pHFucT2) and E. coli (pHFucT2 + pG-KJE3) were measured. When pHFucT2 plasmid was expressed alone in E. coli, the specific activity of crude extract was 0.015 U/mg, whereas when it was coexpressed with pG-KJE3 that encoded DnaK, DnaJ, GrpE, GroES, and GroEL, the specific activity was 0.074 U/mg. The co-expression of chaperone proteins resulted in a 5-fold increase in production of soluble fucosyltransferase in E. coli (Table 2). This result shows that soluble FucT2 was more efficiently expressed when it was co-expressed with pG-KJE3.

Table 2.One unit of activity corresponds to the production amount of 2-FL (2'-fucosyllactose) per min with GDP-L-fucose and lactose.

In this study, the co-expression of Hsp70 (DnaK, DnaJ, GrpE) and Hsp60 (GroEL, GroES) chaperone family from pG-KJE3 successfully improved the amount of FucT2 soluble protein and stabilized FucT2. It is suggested that high levels of chaperones can protect nascent polypeptides from proteolytic attack and facilitate production of native protein [19]. This is probably because interaction of DnaK-DnaJ-GrpE with nascent polypeptides can prevent aggregation that is critical in early step of FucT2 folding. And GroEL-GroES protein transit between soluble and insoluble protein fractions and participates positively in disaggregation [10].

Fucosyloligosaccharides, a member of human milk oligosaccharides family, have many biological functions that have made them the target of various researches. In order to promote the study of these compounds, more efficient synthetic techniques are required. Fucosyltransferase is an essential enzyme of fucosyloligosaccharide production. In previous experiment, fucosyltransferase protein from H. pylori 26695 was expressed in recombinant E. coli, but high level expression of recombinant protein often resulted in aggregation and an accumulation of inclusion bodies. Therefore, for soluble expression of FucT2 gene from H. pylori in E. coli, we attempted co-expression of chaperones at reduced temperatures. For this, molecular chaperones were co-expressed with α1,2-fucosyltransferase (FucT2) in E. coli. The pACYC184 vectors having genes for chaperones such as GroEL, GroES, DnaK, DnaJ, and GrpE were transformed into E. coli BL21 (DE3) star harboring pHFucT2 including FucT2 gene. The transformant was cultured in LB medium and SDS-PAGE analysis showed that 5 chaperones were successfully expressed and the soluble fraction of FucT2 was also increased. HPLC analysis showed that the co-expression of chaperone proteins resulted in a 5-fold increase in total activity of soluble fucosyltransferase in E. coli. In conclusion, this FucT2 expression system can be used as a useful tool for synthesis of fucosyloligosaccharides.