Difference between revisions of "Part:BBa K5004002"

Latest revision as of 12:15, 12 October 2023

a sophisticated enzyme composed of glutathione synthetase GshF and PepG

We have embarked on the development of a sophisticated enzyme composed of glutathione synthetase GshF and PepG. GshF is proficient in catalyzing the synthesis of glutathione, safeguarding hair cells against free radical harm and shielding melanocytes from oxidative damage. On the other hand, PepG is a concise peptide with a composition of 11 polypeptides abundant in cysteine, enhancing the disulfide bonding in keratin and consequently fortifying hair elasticity and strength. To effectively segregate these two proteins, we integrated Thrombin cleavage sites between GshF and PepG. Our research began with examining the activity of GshF in isolation, followed by the construction of engineered Escherichia coli that overexpresses GSHF-PEPG. Practical tests were subsequently performed on hair specimens.

Glutathione is a tripeptide composed of glutamic acid, cysteine, and glycine. In living organisms, glutathione is synthesized through the consecutive action of two enzymes: Glutamate-cysteine ligase (GCL): This enzyme catalyzes the binding of glutamic acid and cysteine to form γ-glutamylcysteine. This is the first step in glutathione synthesis and is typically the rate-limiting step. Glutathione synthetase (GS): This enzyme catalyzes the binding of γ-glutamylcysteine and glycine to form glutathione.

We use a special bifunctional enzyme known as glutathione synthase (GshF), which is derived from the thermophilic streptococcus bacterium Streptococcus thermophilus. This enzyme possesses both GCL and GS activities within a single multifunctional enzyme, allowing it to catalyze these two reactions consecutively.

PepG is a cysteine-rich peptide that is short enough to penetrate the hair's cortex and thus be able to form new disulfide bonds. The decapeptide is small in size (2.72kDa- because PepG exists primarily in dimer form).

Usage and Biology

To produce glutathione, we overexpress GshF in E. coli. Specifically, we used pLac promoter (Lactose promoter) to express gshF in pSB1A3 plasmid, and the recombinant plasmid was transformed into E. coli BL21.

Figure 1 Design of genetic circuit for gshF overexpression.

Figure 2 Gel electrophoresis of the gshF.

To measure the production of Glutathione (GSH), the engineered strain was resuspended in LB medium to an OD600 of 0.1 and incubated at 37°C for 2 hours. The GSH content was determined using a reduced form glutathione (GSH) detection kit, and the results are shown in Figure 3A. Initially, the strain was resuspended in LB medium to an OD600 of 0.1 and incubated at 37°C. Samples were taken at 4, 6, 8, and 12 hours to measure OD600 and assess the impact of GshF on bacterial growth. The results are shown in Figure 3B.

Additionally, different amounts of glutathione were added, and samples were taken at 4, 6, 8, and 12 hours to measure OD600 and assess the impact of glutathione on bacterial growth. The results are shown in Figure 3C. Furthermore, we investigated the effect of adding amino acid precursors (20 mM L-glutamic acid, L-cysteine, and glycine) on GSH production for 2 hours, and the results are shown in Figure 3D.

Figure 3 Experimental results related to GshF.

To determine the enzyme activity of GshF, we cloned its coding gene sequence into pET28a vector and transformed it into E. coli BL21. The engineered bacteria were then cultured overnight in LB medium, and 0.5 mM IPTG was added for induction. The next day, 1 g of bacteria was collected by centrifugation and resuspended in PBS (pH 7.4). The cells were then sonicated (150 W, 1 s on, 3 s off, for a total of 20 minutes) to obtain cell lysate. The reaction mixture in 10 mL PBS contained 20 mM MgCl2, 20 mM ATP, 20 mM L-glutamine, 20 mM L-cysteine, and 20 mM glycine. After incubating at 37°C for 1 hour, the amount of reduced glutathione (GSH) was determined using a GSH assay kit, as shown in Figure 3E. The effect of oxygen on GSH production was also tested (2 hours), as shown in Figure 2F, by culturing the engineered bacteria in a CO2 incubator with O2 concentration adjusted to 0%, 20%, and 30%. After incubating at 37°C for 2 hours, the amount of GSH was measured using a GSH assay kit. The results indicated that the engineered strain significantly enhanced GSH production, with the highest yield at an O2 concentration of 20% （Figure 3F）. The primary component of hair is a protein called keratin, specifically in the form known as α-keratin. Keratin contains numerous chemical bonds called disulfide bonds, which are crucial in determining the shape, elasticity, and strength of hair. PepG is a short peptide rich in cysteine residues, which can bind to keratin, participating in the formation and repair of disulfide bonds, making hair straighter and softer (Figure 4). Previous approaches involved chemical synthesis to obtain PepG (Cruz, Célia F., et al). We intend to use microorganisms to produce this peptide. This is because microorganisms possess certain characteristics that make them well-suited for protein and peptide production: they can grow rapidly and yield high quantities. These microorganisms can automatically secrete the produced peptides, which is very convenient because it means we can easily collect the peptide products from the culture medium. However, it was previously confirmed that PepG could not be efficiently expressed in Escherichia coli due to its small size (iGEM19_Manchester).

Figure 4. The stucture of PepG, which full of cystine residues (Pink lable). This image derived from Part:BBa K2906100 - parts.igem.org)

We aim to express it in E. coli using synthetic biology methods. To achieve this, we have innovatively fused pepG with Glutathione synthetase (GshF) for co-expression, with a Thrombin cleavage site linking the two gene segments（Figure 5）. Thrombin, known for its strong sequence-specific cleavage and high hydrolysis efficiency, is widely used in genetic engineering product development. One of its applications is as a protease tool for the specific cleavage of recombinant fusion proteins. The optimal cleavage site for thrombin is X4-X3-P-R[K]-X1'-X2', where X4 and X3 are hydrophobic amino acids, and X1' and X2' are non-acidic amino acids. The recognition site we are using is L-V-P-R-G-S.

Figure 5. Fusion expression of PepG and GshF proteins. *tcs：Thrombin cleave site. (A) Gel electrophoresis of the GshF-pepG. (B) PepG and GshF are linked through a Thrombin site. (C) plasmid map. The utilized DNA Marker is DL2000, which was procured from Takara in Japan. The bands, ordered from highest to lowest, correspond to DNA fragment sizes of 2000 base pairs (bp), 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp, respectively.

Characterization

The fusion expression gene was synthesized and cloned into pET28a. Subsequently, E. coli BL21 was used as the host to express the fusion protein of GshF and PepG. The engineered bacteria were cultured overnight in LB medium. 0.5 mM IPTG was added for induction. The bacteria sediment was collected by centrifugation the next day and resuspended in 20 mM Tri-HCl buffer (containing 150 mM NaCl, pH 8.0), followed by sonication (150 W, 1s on, 3s off, for a total of 20 minutes) to obtain crude enzyme extract. The protein concentration of the crude enzyme extract was determined using the Bradford assay kit (Solarbio, China).

At 25°C, 100 μg of crude enzyme extract was digested with 2U thrombin for 4 hours. The digestion products were mixed with 20 mM MgCl2, 20 mM ATP, 20 mM L-glutamine, 20 mM L-cysteine, and 20 mM glycine, and incubated at 37°C for 1 hour. The content of reduced glutathione (GSH) was measured using a GSH detection kit. The results showed a significant increase in GSH content in the engineered bacteria (Figure 6A).

Figure 6 Fusion expression of GshF-PepG and thrombin cleavage efficiency.

Subsequently, we proceeded with hair repair using hot-permed hair. Hair was washed with a 0.5% SDS solution, continuously stirred for one hour, and then allowed to air dry. Following this, a portion of the hair sample was secured with adhesive, curled with a clamp, and forcibly straightened to ensure that the hair remained free of any distortion. Afterward, the hair was incubated with the aforementioned cleavage products at 37°C for one hour. Following treatment, the hair was rinsed with distilled water and allowed to air dry naturally. As illustrated in Figure 6B, our finding demonstrated that the cleavage products were successful in restoring the quality of hair.

Potential application directions

The future construction of this engineered strain can help eliminate free radicals, reduce oxidative damage, and protect cells from oxidative stress, playing an antioxidant role. Glutathione has a regulatory effect on the immune system, enhancing the function of immune cells and promoting normal immune responses. It can also be used in industries such as food, pharmaceuticals, and cosmetics. Due to its multiple functions and potential for wide-ranging applications, glutathione has attracted significant attention in scientific research and product development. This project not only opens up avenues for sustainable peptide production using microorganisms but also provides a promising approach to hair care and repair. It's worth noting that these cleavage products are produced by microorganisms, making them a sustainable solution that can be scaled up for hair repair. In comparison to traditional hair repair methods that often rely on harmful chemicals, our approach reduces the dependence on such substances, thereby lowering potential harm to both hair and scalp. This research contributes to improving overall hair health, making hair smoother, softer, and more manageable—particularly beneficial for those with damaged hair or in need of specialized care

References

Ask, Magnus, et al. "Engineering glutathione biosynthesis of Saccharomyces cerevisiae increases robustness to inhibitors in pretreated lignocellulosic materials." Microbial cell factories 12.1 (2013): 1-10. Cruz, Célia F., et al. "Changing the shape of hair with keratin peptides." RSC advances 7.81 (2017): 51581-51592.

Sequence and Features