Part:BBa_K4990005
Auto cleaving linker A
TO KNOW ABOUT IT!
In the wake of advancements in molecular biology, numerous proteins and peptides with active functions have been discovered or invented for pharmaceutical applications. Recombinant technology, characterized by its high expression levels and ease of operation, has been extensively applied in biomedicine and related fields. To obtain multi-target, multifunctional active proteins, it is requisite to link and fuse two or more proteins with known functions. This method of obtaining bifunctional or multifunctional fusion proteins has become one of the new approaches for developing new drugs and researching bioproducts, especially widely used in the preparation of bispecific single-chain variable fragments (scFv) or antibody-drug conjugates[1-5].
Fusion proteins are principally composed of two parts: the functional protein and the linker peptide. The functional protein is the original protein intended for fusion, typically with known structure and function, posing no issues in selection. However, due to the significance of the linker peptide in the overall structure of the fusion protein, its selection and design require thoughtful research to ensure the overall activity of the fusion protein remains unchanged[2]. Consequently, research on linker peptides has gradually come into focus.
What is it?
The three-dimensional structure below illustrate a linker peptide used by us. It is explicit that the linker peptide is composed of simple α-helices. This linker peptide is commonly used as a rigid linker peptide and is widely incorporated into the design of fusion proteins.
Altering the binding interaction of antimicrobial peptides with bacterial cell membranes plays a crucial role in modulating the antibacterial activity of those peptides. Y. Yang et al[6]. substituted Glu16, Asp26, and Glu36 with Gln16, Asn26, and Gln36 respectively, causing the electrostatic charge to increase from +5.8 to +9.0. Such modification elevated the positive charge level of LL-37 without altering its spatial structure. Ga-gnon et al[7]. found that the length of the peptide chain is also related to its antimicrobial activity. Given the same peptide chain length, antimicrobial peptides with longer chains and more positive charges have better antibacterial effects.
Therefore, we anticipate that the linkers at both ends of the bactericidal peptide should not reduce the activity of the antimicrobial peptide due to electrostatic interactions. To avoid positioning negatively charged amino acids near the bactericidal peptide, we designed two types of linkers that allow positively charged lysine to enclose the bactericidal peptide. This is advantageous for designing fusion proteins while ensuring bactericidal activity.
As what is illustrated below, the two lysine residues enclose the red bactericidal peptide, concentrating the positive charge and enhancing the anti-bacteria activity.
What can it do?
BBa K4990005: EAAAK
Property：Rigid Linker
Rigid linker peptides are composed of amino acid residues that readily form stable secondary structures. In many instances, due to their capacity to form such stable secondary structures, flexible linker peptides can more effectively separate functional domains while maintaining their individual functionalities. The spatial separation of functional domains becomes crucial for the stability and bioactivity of fusion proteins when employing rigid linker peptides [8]. Research by George et al. [9] suggests that many natural rigid linker peptides adopt an α-helical structure. The most frequently used rigid α-helical linker peptide in recombinant fusion proteins is (EAAAK)n where n≤6. Owing to its internal hydrogen bonds and a compact backbone, the α-helix is rigid and stable. Experiments by Minsup et al. [10], using flexible (GGGGS) and rigid (EAAAK) linker peptides derived from White starflower beetle and beet armyworm antimicrobial proteins respectively, demonstrated that fusion proteins linked with rigid peptides showed significantly enhanced antibacterial activity compared to their parent proteins. In contrast, fusion proteins with flexible peptides did not exhibit this effect, suggesting that rigid linker peptides can effectively separate functional domains, resulting in proteins with activities surpassing those of the parent proteins. In another study, Guo et al. [11] employed flexible (GGGGS)n (n=1-3) and rigid (EAAAK)n (n=1-3) linker peptides to connect xylanase and mannanase. Comparatively, fusion enzymes linked with the rigid peptide exhibited superior thermal stability. Arai et al. [12] initially designed rigid linker peptide sequences A (EAAAK)n (n=2-5) based on empirical knowledge. This linker peptide adopts an α-helical conformation stabilized by salt bridges between the glutamate and lysine residues. Guo et al. [11] applied these linker peptides for the fusion of mannanase and xylanase, resulting in bi-functional enzyme activities. As the linker length increased, the catalytic efficiency of the fusion enzyme continuously improved. However, when repetitions reached four times, the catalytic efficiency started to decrease, suggesting that adjusting the repetition number of EAAAK can control the distance between structural domains.
In our designed fusion proteins, we extensively utilized this type of rigid self-hydrolyzing linker peptide. We constructed targeting peptides for cancer cell lysis, Fn-targeting peptides, and dual-targeting peptides. We hope that while maintaining the fusion protein's structural domain in an α-helical conformation, it possesses a self-hydrolyzing function, releasing the cytotoxic peptide portion, enabling it to exert its lethal effect.
How does it work?
Wu et al. [11] found that through their research on chitosan-fused rigid linker peptides, that rigid linker peptides exhibit self-cleaving properties under specific conditions. At pH=6-7, cleavage occurs. It's hypothesized that the EAAAK amino acid arrangement can form a stable hydrophilic α-helical structure. The forces at play can be attributed to the Glu-...Lys+ salt bridges between Glu and Lys. Hydrogen bonds are pivotal in forming these salt bridges, so pH greatly influences the stability of the salt bridges. At neutral pH, the strength of salt bridges is at its weakest, making cleavage more likely. Conversely, in high salt environments, the ionic strength can stabilize these bridges, preventing cleavage [12].
Reference
[1]Tao, L., Gao, M., & Zhou, H. (2015). Research progress on novel antibody-chemotherapy drug conjugates. Journal of Pharmaceutical Biotechnology, 22(3), 253-258.
[2] Gustavsson M，Lehtio J，Denman S，et al． Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in pichia pastoris［J］． Protein Eng，2001，14（9）:711-715.
[3] Wang SH，Zheng CJ，Liu Y，et al． Construction of multiform scFv antibodies using linker peptide［J］． J Genetics and Genomics，2008，35:313-316.
[4] Zhang JH，Yun J，Shang ZG，et al． Design and optimization of a linker for fusion protein construction［J］． Progr Natur Sci，2009，19: 1197-1200.
[5] Shan D，Press OW，Tsu TT，et al． Characterization of scFv-Ig constructs generated from the Anti-CD20 mAb 1F5 using linker peptides of varying lengths［J］.J Immunology，2014，162:6589-6595.
[6]Li, J., & Wang, C. (2015). Design of linker peptides and their application in fusion proteins. Journal of Food and Biotechnology, 1121-1127.
[7]Yang, Y., Ge, X., Liu, Y., et al. (2006). Fusion expression and antibacterial activity of modified human LL-37 antimicrobial peptide. Journal of the Fourth Military Medical University, 27(11), 1014-1017.
[8]Gagnon MC，Strandberg E，Grau ‐Campistany A，et al. Influenceof the length and charge on the activity of α‐helical amphipathicantimicrobial peptides［J］. Biochemistry，2017，56（11）：1680-1695.
[9]George R A，Heringa J. An analysis of protein domain linkers：their classification and role in protein folding [J]. Protein Engineering，2003，15（11）：871-879.
[10]Lee M，Bang K，Kwon H，et al. Enhanced antibacterial activity of an attacin-coleoptericin hybrid protein fused with a helical linker[J]. Molecular Biology Reports，2013，40（6）：3953-3960.
[11]Guo N，Zheng J，Wu L，et al. Engineered bifunctional enzymes of endo-1，4-β-xylanase/endo-1，4-β-mannanase were constructed for synergistically hydrolyzing hemicellulose[J]. Journal of Molecular Catalysis B：Enzymatic，2013，97：311-318.
[12]Arai R，Wriggers W，Nishikawa Y，et al. Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering[J]. Proteins：Structure，Function，and Bioinformatics，2004，57（4）：829-838.
[13]Wu Y J, Fan C Y, Li Y K. Protein purification involving a unique auto-cleavage feature of a repeated EAAAK peptide[J]. Journal of Chromatography B, 2009, 877(31): 4015-4021.
[14]Marqusee S, Baldwin R L. Helix stabilization by Glu-... Lys+ salt bridges in short peptides of de novo design[J]. Proceedings of the National Academy of Sciences, 1987, 84(24): 8898-8902.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
iGEM24_Jiangnan-China
Group: iGEM24_Jiangnan-China
Author: Ke Tong, Yueheng Niu, Hanzhi Weng, Zhiyu Shen
Summary: We chose the commonly used linker G4S (BBa_K5157012), EAAAK (BBa_K4990005, from iGEM23_CPU-CHINA), and newly uploaded 10A (BBa_K5157015), AG (BBa_K5157014), and SLE (BBa_K5157016) from the region of the endogenous spacer sequence region of PETase and Tr (BBa_K5157013) originated from Trichoderma reesei [2] as linkers to characterization.
Usage and Biology
Linker as a fragment connecting enzyme and short peptide is categorized into flexible linker, rigid linker and cleavable linker. The selection and design of linker can affect the binding affinity between enzyme and short peptide, and may also provide many other advantages for fusion protein production, such as improving biological activity and increasing expression [1].
Therefore, we tried to replace the linker to optimize the spatial conformation between the enzyme and the short peptide, reducing the mutual influence between the two component proteins, thus improving the substrate catalytic efficiency as well as expression of the fusion protein.
We chose the commonly used linker G4S (BBa_K5157012), EAAAK (BBa_K4990005, from iGEM23_CPU-CHINA), and newly uploaded 10A (BBa_K5157015), AG (BBa_K5157014), and SLE (BBa_K5157016) from the region of the endogenous spacer sequence region of PETase and Tr (BBa_K5157013) originated from Trichoderma reesei [2] as linkers to construct fusion protein.
Molecular construction
The fusion protein gene was synthesised and cloned into plasmid pET-21b（with T7 promotor BBa_K4790075, lac operator BBa_K4790078, RBS BBa_K4790079, and T7 terminator BBa_K4790076) to construct recombinant plasmid pET21b-PETase-linker-70-1-C-His tag.
Protein expression
The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) for expression and cultured in a 50 mL flask to express sufficient fusion protein.
The enzyme activity of fermentation supernatant and supernatant of cell fragmentation was measured by continuous spectrophotometry method. The reaction system was 1500 μL including 30 μL 50 mmol/L pNPB, 30 μL crude enzyme liquid and 1440 μL 100 mmol/L pH 8.0 phosphate buffer. The generation rate of p-nitrophenol during 60 s was recorded at the wavelength of 405 nm. One enzyme activity unit (U) was defined as the amount of enzyme required to hydrolyze pNPB producing 1 μmol p-nitrophenol during 60 s at 60 °C.The sum of the enzyme activities of the fermentation supernatant and supernatant of cell fragmentation was recorded as total enzyme activity.
Molecular dynamics simulation
Molecular dynamics simulations show that the G4S, Tr, and AG linkers are flexible in the fusion protein, while the SLE, EAK, and 10A linkers function as rigid linkers in the fusion protein (Fig. 1).
Fig. 1. Molecular dynamics simulations.
(a). RMSF analog; (b). RMSD analog.
Linker optimization
We constructed fusion proteins by replacing flexible linker G4S with flexible linker Tr, AG and rigid linker SLE, EAAAK, 10A, respectively, then measured enzyme activity after fermentation. As shown in Fig. 2, the highest fusion protein activity was constructed via G4S in the flexible linker, while in the rigid linker, it was SLE. Therefore, we choose to replace the flexible linker G4S with the rigid linker SLE for linker optimization.
Fig. 2. Linker optimization.
We hope that our results of characterizing different linker properties will help other iGEM teams in choosing linkers.
References
[1] Chen X, Zaro J L, Shen W C. Fusion protein linkers: property, design and functionality [J]. Advanced Drug Delivery Reviews, 2013, 65(10): 1357-1369.
[2] Dai L, Qu Y, Huang J W, et al. Enhancing PET hydrolytic enzyme activity by fusion of the cellulose-binding domain of cellobiohydrolase I from Trichoderma reesei [J]. Journal of Biotechnology, 2021, 334: 47-50.
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