TARS Human, Sf9

What is TARS Human produced in Sf9 cells and what are its key characteristics?

TARS Human Recombinant produced in Sf9 insect cells is a glycosylated polypeptide chain with a molecular mass of 83,312 Dalton. It's expressed with a C-terminal 6xHis tag to facilitate purification and is typically prepared through proprietary chromatographic techniques. As a member of the aminoacyl-tRNA synthetase family, TARS plays a crucial role in protein biosynthesis by charging tRNA molecules with threonine amino acids .

The recombinant protein has several synonyms in the literature, including threonyl-tRNA synthetase cytoplasmic, threonine-tRNA ligase, ThrRS, MGC9344, and PL-7. It has EC classification 6.1.1.3 (threonine-tRNA ligase activity) .

Why is the Sf9 insect cell system preferred for expression of TARS Human?

The Sf9 insect cell expression system offers several advantages for the production of complex mammalian proteins like TARS:

• Post-translational modifications: Sf9 cells can perform many eukaryotic post-translational modifications including glycosylation, which is important for TARS structure and function

• High expression levels: The baculovirus expression system in Sf9 cells typically yields higher protein quantities compared to mammalian cell systems

• Proper protein folding: The insect cell environment often allows for correct folding of complex human proteins

• Scale-up potential: Sf9 cultures can be maintained in suspension at densities of 1-5 × 10^6 cells/ml, facilitating larger-scale protein production

Maintaining Sf9 cells in optimal conditions (log-phase growth, 27°C, shaking at approximately 135 rpm) is critical for successful expression .

What are the optimal storage conditions for TARS Human, Sf9 to maintain stability?

TARS Human, Sf9 requires specific storage conditions to maintain stability and activity:

The protein is typically supplied in a stabilizing buffer consisting of 20mM HEPES (pH 8.0), 200mM sodium chloride, and 20% glycerol. Multiple freeze-thaw cycles should be strictly avoided as they can significantly compromise protein integrity and activity .

How should Sf9 cells be maintained for optimal TARS expression?

Successful expression of TARS in Sf9 cells requires rigorous cell culture maintenance:

• Maintain cells strictly in log-phase growth for approximately 15 generations

• Optimal cell density should be kept between 1-5 × 10^6 cells/ml

• Culture at 27°C with shaking at 135 rpm (~1×g) using appropriate platform shakers

• Monitor cell morphology regularly - healthy Sf9 cells should be round in shape, uniform in size, and should not form clumps

• Avoid overgrowth as it leads to decreased protein expression

• Watch for morphological changes such as swelling (accumulation of tetraploid cells) or clumping (environmentally-stressed cells), which indicate that a new culture should be initiated

What is the recommended protocol for baculovirus generation for TARS expression?

The baculovirus expression system for TARS production typically follows this established workflow:

• P0 Virus Generation:

  • Transfect 1.2 μg recombinant bacmid DNA into Sf9 cells using Cellfectin II transfection reagent

  • Prepare transfection mixture by combining bacmid DNA in transfection medium (Solution A) with Cellfectin II in transfection medium (Solution B)

  • Add transfection mixture dropwise to 2.5 ml of Sf9 cells (1 × 10^6 cells/ml)

  • Incubate with shaking (300 rpm, 27°C) for 4 days

• P1 Virus Generation:

  • Add 170 μl of P0 stock to 4 ml of fresh Sf9 cells (1 × 10^6 cells/ml)

  • Incubate with shaking (300 rpm, 27°C) for 4 days

  • Remove cells by centrifugation (700×g, 10 min)

  • Store supernatant (P1 virus) at 4°C for up to 6 months

  • Archive small volumes at -80°C for long-term storage (noting ~1 log drop in viral titer upon thawing)

• Protein Expression:

  • Infect cells at 2 × 10^6 cells/ml with P1 virus at an MOI of 5 IU/cell

  • Calculate virus volume using: (Total cells × desired MOI) ÷ Viral titer

  • Incubate with shaking (135 rpm, 27°C) for 48 hours

  • Harvest cells and verify expression

How can post-translational modifications of TARS in Sf9 cells be characterized?

Characterizing post-translational modifications (PTMs) of TARS expressed in Sf9 cells requires a multi-technique approach:

• Phosphorylation Analysis:

  • Use phosphoprotein-specific staining methods after SDS-PAGE

  • Employ mass spectrometry to identify specific phosphorylation sites

  • Use phosphorylation-specific antibodies for Western blot detection

  • Apply in vitro kinase assays to determine potential phosphorylation sites

• Glycosylation Analysis:

  • Conduct lectin blotting to detect and characterize glycan structures

  • Employ enzymatic deglycosylation followed by mobility shift analysis

  • Use mass spectrometry for detailed glycan profiling

Researchers should note that while Sf9 cells perform many mammalian-like PTMs, the glycosylation patterns differ from human cells, typically producing simpler, high-mannose type glycans rather than complex mammalian glycans .

The relevance of phosphorylation in particular has been highlighted in studies of other proteins expressed in Sf9 systems, where protein functionality can be significantly influenced by phosphorylation status .

How does TARS autoantigenicity relate to its structure and function in research applications?

TARS is recognized as an autoantigen (PL-7 antibody target) in a subset of patients with polymyositis and dermatomyositis. This autoantigenicity has important implications for research:

• Structure-Function Relationships:

  • Epitope mapping using recombinant TARS can identify autoantibody binding regions

  • Site-directed mutagenesis of potential epitopes can clarify structure-function relationships

  • Comparison between native and recombinant TARS can reveal conformational epitopes

• Clinical Correlations:

  • Preliminary data suggest PL-7 antibodies (similar to Jo-1 antibodies) indicate an increased risk for lung involvement in myositis patients

  • Pure recombinant TARS from Sf9 systems enables more precise immunological studies than using cellular extracts

• Research Applications:

  • Recombinant TARS can be used to develop diagnostic assays for autoimmune conditions

  • The protein serves as a tool for studying enzymatic mechanisms of aminoacyl-tRNA synthetases

  • Structure-based drug design targeting TARS may lead to novel therapeutic approaches

What are the key considerations when comparing TARS activity from Sf9-expressed protein versus other expression systems?

When comparing TARS activity across different expression systems, researchers should consider:

• Activity Assessment:

  • Standard aminoacylation assays measuring the charging of tRNA^Thr with radiolabeled threonine

  • ATP-PPi exchange assays to measure the first step of the aminoacylation reaction

  • Enzyme kinetics (Km, Vmax, kcat) comparison between different sources

• System-Specific Differences:

  Expression System	Advantages	Limitations
  Sf9/Baculovirus	Higher yield, eukaryotic PTMs	Insect-type glycosylation patterns
  E. coli	Simple, cost-effective	Limited PTMs, folding issues
  Mammalian cells	Native-like PTMs	Lower yield, higher cost
  Cell-free systems	Rapid expression	Limited PTMs, lower activity

• Functional Considerations:

  • The His-tag on recombinant TARS may affect activity and should be considered in experimental design

  • Buffer conditions significantly impact activity (optimal: 20mM HEPES pH-8, 200mM NaCl, 20% glycerol)

  • Ensure complete removal of any phosphatase inhibitors when studying TARS regulation by phosphorylation

What quality control measures should be implemented for TARS Human, Sf9 preparations?

Rigorous quality control for TARS Human, Sf9 preparations should include:

• Purity Assessment:

  • SDS-PAGE analysis (TARS purity should exceed 90%)

  • Western blot using anti-His antibodies to confirm identity

  • Mass spectrometry to verify molecular weight (expected: 83,312 Dalton)

• Functional Testing:

  • Aminoacylation activity assays using purified tRNA^Thr

  • ATP consumption assays as a surrogate for enzymatic activity

  • Thermal stability assessments to confirm proper folding

• Contaminant Analysis:

  • Endotoxin testing (especially important for immunological studies)

  • Host cell protein (HCP) analysis to detect Sf9-derived contaminants

  • DNA contamination assessment

Researchers should establish acceptance criteria for each parameter based on their specific application requirements .

How can researchers troubleshoot low TARS expression yields in Sf9 cells?

When facing low TARS expression yields, consider the following troubleshooting approaches:

• Viral Quality Issues:

  • Verify viral titer using flow cytometric analysis of gp64 expression

  • Ensure proper storage of viral stocks (4°C for short-term, -80°C for long-term)

  • Prepare fresh P1 virus if titer has decreased significantly

• Cell Culture Problems:

  • Check cell viability and morphology before infection (should be >95% viable)

  • Ensure cells are in mid-log phase (not overconfluent or stressed)

  • Verify cell density at infection (optimal: 2 × 10^6 cells/ml)

  • Monitor for mycoplasma or other contamination

• Expression Conditions:

  • Optimize MOI (try range from 1-10 IU/cell)

  • Adjust harvest timing (try 48-72 hours post-infection)

  • Test different temperature conditions during expression

  • Evaluate different cell lysis methods to improve protein recovery

What strategies can address protein aggregation and solubility issues with TARS Human, Sf9?

Protein aggregation and solubility challenges with TARS can be addressed through:

• Buffer Optimization:

  • Screen different pH conditions (typically pH 7.5-8.5 works best)

  • Test various salt concentrations (150-300mM NaCl range)

  • Include stabilizing agents (glycerol 10-20%, low concentrations of reducing agents)

• Purification Strategies:

  • Employ step-wise elution during affinity chromatography

  • Consider size exclusion chromatography to separate aggregates

  • Add low concentrations of detergents during initial purification steps

  • Perform purification at 4°C to minimize aggregation

• Storage Considerations:

  • Determine optimal protein concentration for storage (typically 1-5 mg/ml)

  • Test flash-freezing in liquid nitrogen versus slow freezing

  • Evaluate addition of cryoprotectants beyond glycerol

  • Consider lyophilization for very long-term storage needs

How can TARS Human, Sf9 be used to study protein-protein interactions in translation machinery?

TARS Human produced in Sf9 cells provides an excellent tool for studying protein-protein interactions within the translation machinery:

• Co-Immunoprecipitation Studies:

  • Utilize the His-tag for pull-down experiments with potential interaction partners

  • Perform reciprocal co-IP experiments to confirm specific interactions

  • Use cross-linking approaches to capture transient interactions

• Structural Biology Applications:

  • The high purity of Sf9-expressed TARS enables crystallization trials

  • Cryo-EM studies can visualize TARS within larger translation complexes

  • NMR studies can map interaction interfaces at the residue level

• Functional Interaction Studies:

  • Reconstitute translation initiation/elongation complexes in vitro

  • Perform competition assays to map binding sites

  • Use mutagenesis to disrupt specific interaction interfaces

What role does phosphorylation play in TARS function and how can this be studied using the Sf9-expressed protein?

Phosphorylation can significantly impact TARS function, and Sf9-expressed protein provides an ideal system to study this:

• Phosphorylation Analysis:

  • In vitro kinase assays can identify which kinases phosphorylate TARS

  • Mass spectrometry can map specific phosphorylation sites

  • Phosphomimetic mutations (S/T to D/E) can simulate constitutive phosphorylation

  • Phosphorylation-resistant mutations (S/T to A) can prevent modification

• Functional Impact:

  • Compare enzymatic activity of phosphorylated versus non-phosphorylated TARS

  • Analyze how phosphorylation affects protein-protein interactions

  • Study subcellular localization changes induced by phosphorylation

• Phosphatase Regulation:

  • Research indicates that protein phosphatases like PP2A can dephosphorylate certain tRNA synthetases

  • The equilibrium between kinase and phosphatase activities may regulate TARS function

  • Phosphatase inhibitors like okadaic acid can be used to maintain phosphorylation states for functional studies

How can TARS Human, Sf9 be utilized in studying autoimmune disorders like polymyositis and dermatomyositis?

TARS Human from Sf9 systems provides valuable tools for autoimmunity research:

• Autoantibody Detection Systems:

  • Develop ELISA-based diagnostics using purified TARS

  • Create addressable antigen arrays for multiplex autoantibody profiling

  • Improve sensitivity and specificity of existing PL-7 antibody detection methods

• Epitope Mapping:

  • Generate TARS fragments to localize autoantibody binding regions

  • Perform competition assays with synthetic peptides to identify linear epitopes

  • Use structural information to identify conformational epitopes

• Clinical Correlations:

  • Study associations between anti-TARS antibody titers and disease progression

  • Investigate the preliminary observation that PL-7 antibodies indicate increased risk for lung involvement

  • Compare epitope specificity with clinical manifestations to identify predictive biomarkers

• Mechanistic Studies:

  • Investigate how autoantibodies affect TARS enzymatic activity

  • Study whether autoantibodies alter TARS localization in cells

  • Examine potential molecular mimicry between TARS and pathogen proteins