Recombinant Human GPT2 Protein: Research Guide & Protocol

Recombinant Human GPT2 Protein is a research-grade recombinant enzyme representing the human glutamic pyruvate transaminase 2 isoform, widely used to investigate amino acid catabolism, gluconeogenesis, and mitochondrial alanine metabolism in biochemical and cell-based assays.

Product Name

Recombinant Human GPT2 Protein

SKU

rp374702

Gene / Protein Target

GPT2 (Glutamic Pyruvate Transaminase 2; Alanine Aminotransferase 2)

Protein Function

Catalyzes reversible transamination between alanine and 2-oxoglutarate to form pyruvate and glutamate; mitochondrial isoform involved in amino acid catabolism and gluconeogenesis

Expression System

Recombinant (host system to be confirmed with product datasheet)

Recommended Storage

-20°C or -80°C; avoid repeated freeze-thaw cycles (confirm with datasheet)

Primary Applications

Enzymatic activity assays, Western blot standard, functional studies of amino acid metabolism, gluconeogenesis research

Overview of GPT2 Biology and Research Significance

GPT2, also known as Glutamic Pyruvate Transaminase 2 or Alanine Aminotransferase 2 (ALT2), is the mitochondrial isoform of alanine aminotransferase. It catalyzes the reversible transamination reaction between L-alanine and 2-oxoglutarate (α-ketoglutarate) to produce pyruvate and L-glutamate, utilizing pyridoxal phosphate (PLP) as an essential cofactor.

Unlike the cytosolic isoform GPT1 (ALT1), GPT2 is predominantly localized to the mitochondrial matrix, positioning it at a critical junction between amino acid catabolism, the tricarboxylic acid (TCA) cycle, and gluconeogenesis. This enzyme plays a key role in alanine-glucose cycling and is of particular relevance in tissues with high metabolic activity such as liver, skeletal muscle, and brain.

Research interest in GPT2 has grown considerably due to its reported involvement in cancer cell metabolism, neurological development, and metabolic disorders. Recombinant Human GPT2 Protein provides a well-defined, reproducible tool for in vitro enzymatic studies, inhibitor screening, and structural investigations.

Protein Specifications and Quality Attributes

Recombinant Human GPT2 Protein (SKU: rp374702) is produced as a recombinant protein designed to reflect the native human GPT2 sequence. Researchers should consult the official product datasheet for confirmed specifications including molecular weight, purity grade, endotoxin levels, expression host, and tag information, as these parameters are critical for experimental design.

Key quality attributes to verify on the datasheet include:

• Purity: Typically assessed by SDS-PAGE; pharmaceutical-grade recombinant proteins commonly target ≥90% purity, though exact values should be confirmed.
• Endotoxin Level: Important for cell-based assays; confirm the endotoxin specification from the product certificate of analysis (CoA).
• Biological Activity: Confirm whether enzymatic activity data (e.g., specific activity in nmol/min/mg) are provided on the datasheet.
• Formulation: Buffer composition, carrier protein presence (e.g., BSA), and glycerol content affect downstream assay compatibility.

Always request and review the Certificate of Analysis (CoA) and Safety Data Sheet (SDS) prior to use.

Reconstitution and Storage Protocol

Proper reconstitution and storage are essential to maintain the activity and integrity of Recombinant Human GPT2 Protein. The following general protocol is based on standard best practices for lyophilized or liquid recombinant proteins; always defer to the product-specific datasheet for exact instructions.

Reconstitution (if lyophilized)

1. Briefly centrifuge the vial before opening to consolidate the lyophilized powder.
2. Reconstitute in sterile, high-quality water (e.g., nuclease-free water) or the buffer specified in the datasheet, typically at a concentration of 0.1–1.0 mg/mL.
3. Gently swirl or pipette to dissolve; do not vortex vigorously as this may denature the protein.
4. Allow to equilibrate at room temperature for 5–10 minutes, then place on ice.

Storage

• Short-term (up to 1 week): Store at 4°C if the protein will be used promptly.
• Long-term: Aliquot into single-use volumes and store at -20°C or -80°C to avoid repeated freeze-thaw cycles.
• Addition of a carrier protein (e.g., 0.1% BSA) may improve stability during storage; confirm compatibility with your assay.

Note: Repeated freeze-thaw cycles significantly reduce enzymatic activity. Prepare working aliquots immediately after reconstitution.

Key Applications in Research

Recombinant Human GPT2 Protein is suited for multiple research applications across biochemistry, cell biology, and metabolic research:

• Enzymatic Activity Assays: Use as a positive control or as the active enzyme in kinetic assays measuring transamination activity. Coupled assay systems with lactate dehydrogenase (LDH) or glutamate dehydrogenase (GDH) allow spectrophotometric monitoring of reaction progress.
• Inhibitor Screening: GPT2 is a potential therapeutic target in oncology and metabolic disease. The recombinant protein enables high-throughput or focused inhibitor screening campaigns in vitro.
• Western Blot Standard: Use as a molecular weight marker and positive control in immunoblotting experiments targeting GPT2.
• Protein-Protein Interaction Studies: Investigate binding partners and regulatory complexes involving GPT2 using pull-down assays or co-immunoprecipitation setups with recombinant protein.
• Structural Studies: Support crystallography or cryo-EM studies when sufficient quantities of pure recombinant protein are available.
• Metabolic Flux Studies: Complement isotope-tracing experiments by providing defined enzymatic activity references for alanine-glucose cycling models.

Experimental Tips and Troubleshooting

To maximize experimental success with Recombinant Human GPT2 Protein, consider the following guidance:

Optimizing Enzymatic Assays

• Ensure the reaction buffer pH is optimized for GPT2 activity; alanine aminotransferases generally show optimal activity near physiological pH (7.2–7.6).
• Include pyridoxal phosphate (PLP) in assay buffers if not already present in the protein formulation, as PLP is an obligate cofactor for all aminotransferases.
• Pre-warm substrates and buffers to 37°C before initiating reactions for consistent kinetic data.

Avoiding Common Pitfalls

• Protein aggregation: If turbidity is observed after reconstitution, briefly centrifuge at low speed and use the supernatant.
• Loss of activity: Verify that freeze-thaw cycles have been minimized; always use freshly thawed aliquots.
• Non-specific signal in immunoassays: Optimize antibody concentrations and blocking conditions when using GPT2 protein as a Western blot standard or ELISA antigen.

Controls

Always include appropriate negative controls (e.g., heat-inactivated protein) and positive controls in enzymatic assays to validate assay performance and confirm protein activity upon receipt.

Frequently asked questions

What is GPT2 and why is it important in research?

GPT2 (Glutamic Pyruvate Transaminase 2) is the mitochondrial isoform of alanine aminotransferase, catalyzing reversible transamination between alanine and 2-oxoglutarate. It is important in research because it sits at the crossroads of amino acid catabolism, the TCA cycle, and gluconeogenesis, and has been implicated in cancer metabolism, neurological development, and metabolic disease.

How should I store Recombinant Human GPT2 Protein to maintain its activity?

For long-term storage, aliquot the protein into single-use volumes and store at -20°C or -80°C to prevent activity loss from repeated freeze-thaw cycles. For short-term use within one week, storage at 4°C is generally acceptable. Always consult the product datasheet for formulation-specific storage recommendations.

What cofactor is required for GPT2 enzymatic activity and should it be added to assay buffers?

GPT2 requires pyridoxal phosphate (PLP) as an essential cofactor, as is common to all aminotransferases. If PLP is not already included in the protein formulation, it should be supplemented in the assay buffer to ensure full enzymatic activity. Check the product datasheet to determine whether PLP is present in the supplied formulation.

What assay formats are compatible with Recombinant Human GPT2 Protein?

Recombinant Human GPT2 Protein is compatible with coupled enzymatic assays (e.g., linked to LDH or GDH for spectrophotometric readout), Western blot as a positive control or molecular weight standard, ELISA antigen applications, pull-down and co-immunoprecipitation assays, and in vitro inhibitor screening formats. Confirm assay-specific compatibility with the protein formulation details on the datasheet.

How do I distinguish GPT2 from GPT1 (ALT1) in my experiments?

GPT2 is the mitochondrial isoform and GPT1 is cytosolic; they differ in subcellular localization, tissue expression patterns, and sequence identity. Isoform-specific antibodies are required for immunoassay distinction. In enzymatic assays using the recombinant protein, you are working with a defined isoform, eliminating ambiguity. Refer to published literature for isoform-specific inhibitor selectivity data if relevant to your study.

References

1. Sperringer JE, Addington A, Hutson SM. Branched-chain amino acids and brain metabolism. Neurochemical Research. 2017;42(6):1697–1709. (General aminotransferase biology context; GPT2 specifics should be verified.)
2. Yang RZ, et al. Alanine aminotransferase isoenzymes: molecular cloning and quantitative analysis of tissue expression in rats and serum elevation in liver toxicity. Hepatology. 2009;49(2):598–607.
3. Aladdin Scientific Product Page: Recombinant Human GPT2 Protein, SKU rp374702. (Internal reference; consult official datasheet for confirmed specifications.)

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.