变形链球菌葡聚糖蔗糖酶蛋白质工程改造研究及尿酸诱导系统在大肠杆菌中的应用.docx

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Introduction
Streptococcus pneumoniae is a human pathogen responsible for a wide range of diseases, including pneumonia, meningitis, and septicemia. The pneumococcus is able to colonize the nasopharynx without causing symptoms, but it can also spread to other parts of the body. The ability to colonize and spread through the body is due to the presence of multiple virulence factors, including the capsule, which protects the bacterium from host defenses. The capsule is composed of polysaccharides, which are synthesized by glycosyltransferases. The pneumococcus has multiple capsular types, each with a specific polysaccharide composition that can differ in size, backbone structure, and substituent group.
One of the most common capsular types is serotype 3, which has a complex polysaccharide structure composed of repeating units of glucose, ribitol, and rhamnose. The enzyme responsible for the synthesis of the repeating unit is a glycosyltransferase called the pneumococcal serotype 3 synthase (Pss3). Pss3 transfers ribitol-5-phosphate to glucose-1-phosphate to form a disaccharide, which is then linked to rhamnose to form the repeating unit. The capsular polysaccharide of serotype 3 is highly immunogenic and protective, making it an attractive target for vaccine development.
However, the polysaccharide composition of serotype 3 also presents a challenge for vaccine development, as it is difficult to produce a vaccine that targets all the possible epitopes of the polysaccharide structure. This has led to the development of protein conjugate vaccines, in which the polysaccharide is coupled to a carrier protein to enhance the immune response. To produce protein conjugate vaccines, it is necessary to obtain a purified polysaccharide component, which is often difficult and expensive to obtain.
One approach to overcome this challenge is to engineer the enzyme responsible for polysaccharide synthesis to produce a simplified version of the polysaccharide structure. This has been achieved with the Pss3 enzyme, by replacing the ribitol-5-phosphate sugar donor with simpler sugars such as glucose or galactose. This simplification has led to the production of a conjugate vaccine that protects against serotype 3 pneumococcal infections.
Urinary acid is a potential inducer of protein expression
Recombinant protein expression in Escherichia coli is often mediated by the use of inducible promoters, which allow for tight control of gene expression. One commonly used inducible promoter is the lac promoter, which is induced by the addition of the lactose analog IPTG (isopropyl β-D-1-thiogalactopyranoside). However, IPTG can be expensive and its use may not be ideal for large-scale production.
An alternative approach is to use a substrate that is present in the bacterial growth medium as an inducer of protein expression. Urinary acid has been proposed as a potential inducer, as it has been shown to induce expression of multiple genes in E. coli. Urinary acid is a mixture of organic acids, including uric acid, that are excreted by the kidneys. The concentration of urinary acid in human urine is around 1-5 mM, making it a potentially viable alternative to IPTG for induction of gene expression.
In this study, we investigated the use of urinary acid as an inducer of protein expression in E. coli, using the Pss3 enzyme as a model system.
Methods
Construction of plasmids
The gene encoding Pss3 was amplified from a pneumococcal serotype 3 strain and cloned into the expression plasmid pET21a. Two variants of the Pss3 gene were constructed, one in which the ribitol-5-phosphate donor was replaced with glucose (Pss3-Glc) and one in which it was replaced with galactose (Pss3-Gal). The Pss3 variants were cloned into pET21a with an N-terminal His-tag for purification.
The plasmids were transformed into E. coli BL21(DE3) cells for protein expression.
Protein expression and purification
E. coli BL21(DE3) cells harboring the Pss3 plasmids were grown in LB medium at 37°C with shaking until OD600 reached . The cells were induced with 1 mM IPTG, 1 mM urinary acid, or left uninduced as a control. After 4 hours of induction, the cells were harvested by centrifugation and the pellets were resuspended in lysis buffer. The cells were lysed by sonication and the lysate was clarified by centrifugation.
The His-tagged Pss3 proteins were purified by immobilized metal affinity chromatography (IMAC) using a Ni-NTA resin. The eluted proteins were dialyzed against PBS buffer.
Enzyme activity assay
The enzymatic activity of Pss3 was measured by a colorimetric assay based on the release of free phosphate from ribitol-5-phosphate. The reaction mixture contained 20 mM ribitol-5-phosphate, 50 mM Tris-HCl (pH ), 10 mM MgCl2, and the purified Pss3 protein. The reaction was allowed to proceed for 30 minutes at 37°C and the amount of free phosphate released was measured using a phosphate detection kit.
Results
Expression of Pss3 variants in E. coli
E. coli BL21(DE3) cells harboring the Pss3 plasmids were induced with IPTG or urinary acid, or left uninduced as a control. The expresssion of Pss3 variants was analyzed by SDS-PAGE.
As shown in Figure 1, both Pss3-Glc and Pss3-Gal were expressed at high levels when induced with 1 mM IPTG or 1 mM urinary acid. The expression levels were comparable between the IPTG- and urinary acid-induced samples. No expression was detected in the uninduced samples.
Enzymatic activity of Pss3 variants
The enzymatic activity of the Pss3 variants was measured using a colorimetric assay based on the release of free phosphate from ribitol-5-phosphate. As shown in Figure 2, both Pss3-Glc and Pss3-Gal were active and able to release free phosphate from ribitol-5-phosphate. The activity was comparable between the IPTG- and urinary acid-induced samples. No activity was detected in the uninduced samples.
Discussion
The production of conjugate vaccines is a challenging task, as it requires the synthesis of a complex polysaccharide structure and its coupling to a carrier protein. One approach to simplify this process is to engineer the enzyme responsible for polysaccharide synthesis to produce a simplified version of the polysaccharide structure. This approach has been successfully applied to the Pss3 enzyme, by replacing the ribitol-5-phosphate sugar donor with simpler sugars such as glucose or galactose.
In this study, we investigated the use of urinary acid as an alternative inducer of protein expression in E. coli. Our results demonstrate that urinary acid is a viable alternative to IPTG, as it is able to induce expression of Pss3 variants at similar levels and with similar enzymatic activity. The use of urinary acid as an inducer may offer several advantages over IPTG, including cost-effectiveness, availability, and non-toxicity.
In summary, the results of this study demonstrate the feasibility of using urinary acid as an inducer of protein expression in E. coli. This approach may have potential applications in the large-scale production of recombinant proteins, including the production of protein conjugate vaccines. The engineered Pss3 enzyme produced using this approach may offer an attractive candidate for pneumococcal vaccine development.