Construction of the expression vector and location analysis of thermotolerant endoglucanase in E. coli

Abstract

To obtain the secreting expression vector, the signal peptide sequence and mature peptide sequence of endoglucanase from BoldItalic KX6 were cloned into the pET28a plasmid. The recombinant vector pET28a/KX6 was transformed BoldItalic Rosetta (DE3), and the transformant was induced by IPTG. The expression products were primarily distributed in the medium fluid of host cell in a soluble form and the activity was higher than that of other fractions. Both location analysis of targeting protein and activity analysis showed that the signal peptide of endoglucanase from BoldItalic KX6 had played a very important role in the secret expression and activity of foreign proteins in the BoldItalic host cell.

Keywords

thermotolerant endoglucanase / expression vector / signal peptide / location analysis

Introduction

The endocellulase (EC 3.2.1.4), the major component of the cellulolytic enzyme system, first attacks on the cellulose polymer by breaking down the internal β-1,4 glucosidic linkages within amorphous regions of cellulose chains (Wulff et al., 2003). Therefore, it plays an important role in degradation of cellulosic biomass (Solingen et al., 2001). A number of cellulase genes from bacteria and fungus have been cloned and also expressed in yeast or Escherichia coli (Qiao et al., 2004; Kim and Lee, 2008). However, no information on endoglucanase gene from Streptomyces xylophagus was found.

Pure, soluble, and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity and ease of isolation or price; hence, recombinant technology is often the method of choice. Recombinant cell factories are constantly employed for the production of protein preparations for downstream purification and processing (Sørensen and Mortensen, 2005). E. coli is a frequently used host, but expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins (Huo et al., 2008).

Here, we attempted a new approach to overcome these obstacles above by a strategy that focuses on controlled expression of target protein in a soluble form through target signal sequence. The main purpose of our study was to obtain a high degree of accumulation of soluble product in the bacterial cell cultures for simplified purification and large-scale production and thus to provide a basis for understanding the secreting mechanism of recombinant proteins in E. coli.

Materials and methods

Strains and plasmids

The vectors pET-28a and pMD18-T-cel (containing the target gene cel) and host strains E. coli JM109 and Rosetta (DE3) were conserved in the Enzyme Engineering Laboratory of Agriculture University of Hebei, Baoding, China.

Construction of recombinant expression vectors

The pair of specific primers was designed according to the cellulase gene sequences from S. xylophagus KX6 (FJ441063): P1: 5′-TCGAATTCGATGCCACGGTTAC-3′ (underlined is the site of EcoRI) and P2: 5′-TTGCGGCCGCTCACGCGGTGCT-3′ (underlined is the site of NotI).

The full-length DNA sequence of endoglucanase gene cel was obtained by PCR amplification using pMD18-T-cel as a template. PCR amplification was done using a Thermal Cycler 100 (USA) in 50 µL reaction volumes for 30 cycles of 94°C for 1 min, 63°C for 1 min, and 72°C for 1 min. The PCR product of about 1200 bp was purified by agarose gel electrophoresis. The PCR products and the expression vector pET-28a were digested by the EcoRI/NotI, respectively, and then ligated in vitro to construct the recombinant plasmid pET-28a/KX6. To verify the gene cel and its signal sequence under the transcriptional control of the T7 promoter, the recombinant plasmid pET-28a/KX6 was sequenced. After the construct was verified, plasmid pET-28a/KX6 was transformed into an expression host for protein production.

Induced expression of recombinant proteins

The recombinant vector pET-28a/KX6 was transformed E. coli Rosetta (DE3) competent cells and the transformants were screened at LB plate containing kanamycin. The different resistance transformants were picked and inoculated into 10 mL NZYCM medium and grown at 30°C in a shaking incubator (250 r·min^-1) for 12 to 16 h. Then, 1% inoculation was conducted into 100 mL NZYCM medium. When the culture grew at 30°C and reached an OD600= 0.4-0.6, IPTG was added to a final concentration of 1 mmol·L^-1 to induce expression. The culture and cell pellet from the different locations of the cell were used to analyze the cellulase activity and solubility.

SDS-PAGE of the TCP

The expression of target genes may be assessed by analysis of total cell protein (TCP) on a SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie blue staining. SDS-PAGE was performed in gel containing 12% (w/v) acrylamide and 0.1% SDS (w/v) using a Tris/glycine buffer system. The protein was stained with 0.1% Coomassie brilliant blue R250 in 7% (v/v) acetic acid in 50% (v/v) methanol solution. Destaining was carried out with 7% acetic acid in 50% methanol.

Location of endoglucanase in the E. coli cells

The location of target protein was performed according to the Manual to pET system. The extracellular fluid from the E. coli engineering strain was extracted by centrifugation at 10000×g for analyzing the solubility and activity. The cell pellet was saved and allowed to drain. After removing as much liquid as possible, the pellet was resuspended thoroughly in 30 mL of 30 mmol·L^-1 Tris-HCl at pH 8.0 and 20% sucrose followed by addition of 60 L of 0.5 mol·L^-1 EDTA at pH 8.0 into the suspension to reach the final concentration of 1 mmol·L^-1, which was stirred slowly using a magnetic stirring bar at room temperature for 10 min. The cells were collected by centrifugation at 10000×g at 4°C for 10 min. After the pellet was thoroughly resuspended in 30 mL of ice-cold 5 mmol·L^-1 MgSO₄, the cell suspension was stirred slowly for 10 min on ice. During this step, the periplasmic proteins were released into a buffer and the cell pellet was ice-stored for further processing of the soluble and insoluble cytoplasmic fractions. Through adding 1 L (25 units) lysozyme per 1 mL BugBuster reagent used for resuspension, rotating mixer at a slow setting for 20 min at room temperature, and centrifuging the mixture at 16000×g for 20 min, the supernatant was gained and placed into a fresh tube for analysis. The pellet may be used to isolate the insoluble cytoplasmic fraction. The final pellet of inclusion bodies was compatible with resuspension in denaturing buffer.

Determination of endoglucanase from various fractions

Enzyme activity was assayed with CM-cellulose as a substrate by measuring the amount of reducing sugar in various fractions according to the dinitrosalicylic acid method of Miller (1959). The reaction mixture contained 0.8 mL of 1% CM-cellulose solution of 200 mmol·L^-1 citric acid-Na₂HPO₄ buffer at pH 5.5 and 0.2 mL appropriately diluted enzyme solution, which was incubated at 50°C for 20 min, and the reaction was stopped by addition of 0.5 mL DNS reagent. The reducing sugar generated was quantified at A540 by adding 3 mL water and using D-glucose as a standard for spectrophotometric estimation. One unit of endoglucanase activity was defined as the amount of enzyme that releases 1 µg reducing sugar per minute under the assay conditions. The cogon red–CMCNa plate (containing 1% CMCNa, 0.05% cogon red, and 2% agar) was also used to determine the endoglucanase activities of expressing proteins. By adding 50 μL sample into the hole (0.6 mm in diameter), which was incubated at 50°C for overnight, the enzyme activity was determined and described by the size of discoloring zone around the hole.

Results

The construction of recombinant vector

The target sequence about 1200 bp with signal sequence was cloned via PCR and then inserted into the pET-28a vector to construct the secreted recombinant expression vector pET28a/KX6. The recombinant plasmid DNA was isolated using standard preparation procedure and then digested with restriction enzymes EcoRI and NotI. The result showed two bands about 5.9 kb and 1.2 kb were found on agarose gel electrophoresis, respectively, which conforms to the size of prediction (Figs. 1 and 2). The sequencing result also suggested the insert sequence and open reading frame are fully accurate.

SDS-PAGE analysis of TCP

After a target plasmid was transformed into E. coli Rosetta (DE3), the expression of the target DNA was induced by the addition of IPTG to a growing culture. The induced cultures were examined for the production of the target protein. The expression of target gene was assessed quickly by analysis of TCP on SDS-PAGE followed by Coomassie blue staining. SDS-PAGE analysis of TCP could be seen in Fig. 3. From Lanes 3 to 9, the target protein was increasingly accumulated along with IPTG induction. The size of expression protein was about 43 KD, the same as putative molecular weight.

Location of endoglucanase in E. coli cells

The target protein may be distributed to different locations of E. coli cells. A small-scale sample of the four fractions in the following medium, periplasm, soluble cytoplasm, and insoluble cytoplasm is analyzed by SDS-PAGE. The results showed the target product was mainly distributed the extracellular fluid as soluble form (Fig. 4, Lane 5) and cytoplasmic fraction as inclusion bodies (Fig. 4, Lane 2). However, a small part of the target protein also was found in the cytoplasm as soluble fractions (Fig. 4, Lane 3) and hardly determined target protein in periplasm (Fig. 4, Lane 4).

The distribution of the target protein suggested the expression product of target gene secreted into the medium fluid; moreover, expression levels right in the culture medium were higher than that in other ones, indicating its contribution to recovering and purifying the target products from the extracellular fluid. In addition, amounts of target proteins also aggregated in the cytoplasmic fraction known as inclusion bodies, and the formation of insoluble inclusion bodies suggested that the high rate of protein synthesis could not export the target protein or folded properly, which led to aggregation in the cytoplasm in a soluble form. Results of this analysis may lead to further optimization of the induction conditions and decreasing the rate of protein synthesis, such as low induction temperatures or growth in the minimal medium tended to increase the percentage of target protein in soluble form. A TCP sample was also analyzed in parallel with the various fractions serving as a control for recovery of the target protein (Fig. 4, Lane 1).

Activity analysis of expression product

The samples from the medium fluid and soluble cytoplasmic fraction showed high specific endoglucanase activities with 534.24 and 127.44 U, respectively, whereas the inclusion bodies could only present 3.78 U, which might have resulted from the retaining soluble cytoplasmic fraction (Table 1). In this study, the cogon red–CMCNa plate was also used to determine the specific endoglucanase activities of expressing protein; the results conformed to that from the spectrophotometry method. The supernatant from medium fraction revealed the largest transparency zone among four fractions, followed soluble cytoplasmic fraction and insoluble cytoplasmic fraction, and could hardly show the bleaching circle around the hole No. 2 poured by the periplasmic fraction (Fig. 5). These results suggested that vector carrying signal peptide sequence was used to export significant amounts of active and soluble target proteins to the extracellular medium for simplifying purification.

Discussion

Recombinant proteins expressed in E. coli are often produced as aggregates called inclusion bodies. In many applications, however, it is desirable to express target proteins in their soluble and active forms.

To obtain active and soluble extracellular proteins, secreted expression vector was constructed to allow the target protein export into the periplasm and even extracellular medium (Lei et al., 2008). For this purpose, many vectors, such as pET-20, pET-22, and pET-40, are designed to carry the signal sequence, but the target proteins will not be desirable for periplasmic localization, and recombinant proteins expressed in E. coli are often produced as aggregates called inclusion bodies. Ikemura et al. (1987) found that the pro-sequence of subtilisin plays an important role in the formation of enzymatically active subtilisin. We assumed the signal peptide sequence of target genes may be contributive to the expression product transportation of the E. coli cells through across-membrane. Based on this idea, we selected pET-28a as expression vector without signal sequence and made the heterologous gene cel and its signal sequence insert the pET-28a vector to form a complete opening reading frame. The recombinant vector pET-28a/KX6 was transformed host cell E. coli Rosetta (DE3) and high-level gene expression products were produced into medium fluid. Obviously, the self-signal sequence plays an important role in secreting the target protein into medium in a soluble and active form.

With each target protein coming from engineering strains, it is important to verify the localization and estimate the yield in the culture medium or cell. In general, with the high expression levels of the pET system, there may exist part of soluble material in the periplasm and most of the insoluble target protein mass in aggregates. Takeshi Yamamoto et al. (1991) reported that chimeric ras protein with OmpF signal peptide expressed in E. coli and OmpF fusion protein was localized in the inner membrane. The target signal sequence may leak into the medium to export proteins to the periplasm with the CBD cenA simplified purification (Weickert et al., 1996). In our experiment, the most target proteins were distributed in medium fluid instead of periplasmic space, which is not consistent with the results from other researches that many recombinant proteins are directed to the periplasm and often end up in the medium through a poorly understand “leakage” phenomenon. Stader and Silhavy (1990) thought that target proteins, in most cases, leaked into the medium due to a damage to the cell envelope rather than true secretion. However, in our study, it is incredible for the mass of target proteins to leak to the extracellular medium unless target proteins secrete into the medium. Therefore, the function of signal peptide sequence of target genes needs to be further studied, which will provide a basis for understanding the target protein secreting mechanism in E. coli.

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