Research Advances in Preparation Technology and Functional Applications of Myk2 Recombinant Protein

Molecular Characteristics and Optimization of Recombinant Expression Systems

As a core regulatory factor of the plant MAPK cascade pathway, preparing Myk2 recombinant protein overcomes challenges in eukaryotic kinase expression within prokaryotic systems. Composed of 621 amino acids (molecular weight ~70.5 kDa), it contains an N-terminal kinase domain (residues 32–302) and a C-terminal regulatory module (coiled-coil domain CCD and auto-inhibitory domain AID). In E. coli systems, codon optimization, low-temperature induction (16°C), and co-expression with molecular chaperones (GroEL/GroES) boost soluble expression to 15–20 mg/L. However, prokaryotic products often lack key phosphorylations (e.g., T312/S316 in the activation loop), requiring in vitro reactivation by MKKK kinases. In contrast, the baculovirus-insect cell system (Sf9 cells) enables native modifications: ① auto-phosphorylation of the activation loop (mass spectrometry confirms >90% phosphorylation); ② O-GlcNAc glycosylation at C-terminal S585; ③ formation of functional dimers (SEC-MALS measures 141 kDa molecular weight). With yields of 5–8 mg/L, purification via Ni-NTA affinity chromatography and size-exclusion chromatography achieves >98% purity (HPLC-verified). A newly developed cell-free wheat germ system synthesizes 1.2 mg/mL active Myk2 within 6 hours, ideal for isotope (15N/13C)-labeled NMR studies.

Functional Verification and Structural Biology Breakthroughs

Functional validation of recombinant Myk2 involves multi-level analysis. In vitro kinase assays show 3.5-fold higher phosphorylation efficiency (kcat/Km=2.8×10⁴ M⁻¹s⁻¹) for substrate MKK4 versus MKK5, attributed to a specific docking interface: SPR determines Kd=38 nM for Myk2 CCD domain (residues 450–500) binding to MKK4’s N-terminus. Cryo-EM structures of the Myk2-MKK4 complex (3.2 Å) reveal three key mechanisms: ① conformation of the D166-K170-E174 catalytic triad in the ATP-binding pocket; ② allosteric effects from activation loop phosphorylation (12° αC helix displacement); ③ hydrophobic network at the CCD-KIM interface (involving L483/V485 and MKK4 F32/L35). Gain-of-function mutants (T312D/S316E) show 5-fold higher specific activity (1200 pmol/min/μg) than wild-type, while kinase-dead mutants (K170M) lose all function. HDX-MS localizes the binding site of small-molecule inhibitor MCIS1 to the junction of the kinase domain and CCD, with IC50=0.8 μM.

Application Research and Technological Innovations

Active recombinant Myk2 provides critical tools for plant stress resistance studies. Phosphoproteomics using recombinant Myk2-treated Arabidopsis protein extracts identifies 22 novel substrates, including stomatal regulator SLAC1 (8-fold enhanced S206 phosphorylation) and redox enzyme RBOHD (dual phosphorylation at S343/S347). An AlphaScreen-based high-throughput platform uses His6-Myk2 as a target to screen 100,000 compounds, yielding allosteric inhibitor Myki-17 (Kd=65 nM), which reduces downy mildew infection area by 85% in tobacco leaves. In synthetic biology, recombinant Myk2 integrates into microfluidic artificial cell systems with FRET biosensors (Myk2-snRK2.6) to monitor drought signaling dynamics (7-fold kinase activity increase within 3 minutes). The latest application involves nanoelectroporating Alexa Fluor 647-labeled Myk2 into plant protoplasts, where single-molecule tracking reveals stress granule localization: 48% of Myk2 aggregates into 200 nm signal hubs within 10 minutes under mannitol stress.

Technical Challenges and Solutions

Current bottlenecks for Myk2 recombinant protein include: ① low activity in prokaryotic expression (only 20% of insect cell systems), addressed by developing E. coli-yeast shuttle vectors with ScFv¹⁰ antibodies to mimic auto-inhibitory domain dissociation; ② poor long-term stability (70% activity loss after 1-week storage at 4°C), resolved by introducing C483S/C520S double mutations for oxidation-stable Myk2 (half-life extended to 4 weeks); ③ difficult complex crystallization, improved 3-fold by fixing Myk2-MKK4 complexes with crosslinker BS³. Future directions include: ① using AI algorithms (e.g., AlphaFold-Multimer) to predict optimal expression conformations; ② developing light-activatable LOV-Myk2 fusion proteins; ③ constructing nanodisc-encapsulated membrane-localized Myk2 to mimic natural environments; ④ establishing single-molecule FRET (smFRET) platforms to analyze kinase conformational dynamics. These innovations will advance Myk2 recombinant protein from basic research to agricultural applications, providing molecular switches for intelligent stress-resistant crop design.

Product Information

Disclaimer: This article partially utilizes artificial intelligence assistance in its creation. If any content involves copyright or intellectual property issues, please let us know and we promise to verify and remove it as soon as possible.

ANT BIO PTE. LTD. – Empowering Scientific Breakthroughs

At ANTBIO, we are committed to advancing life science research through high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) cover a full spectrum of research needs, from general reagents and kits to antibodies and recombinant proteins. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in unlocking scientific mysteries and driving medical progress. Explore our product portfolio today and elevate your research to new heights.