Full workflow solution for recombinant protein expression, purification, and quality control

A one-stop protein preparation solution from host and vector selection, small-scale screening, and culture amplification, to affinity capture, chromatography polishing, buffer exchange and concentration, and quality control.

Experimental Objective

To obtain recombinant proteins that meet the requirements for biochemical, structural, cellular, or immunological research, and to establish a repeatable and scalable expression and purification process.

Scope of Application and Experimental Requirements

  • Host: E. coli, yeast, insect cells, or mammalian cells
  • Requirements: Clearly define domain, tag, post-translational modification, yield, purity, monomer ratio, activity, endotoxin, and sterility requirements
  • Duration: From 3 days to several weeks, depending on construct and process complexity
  • Safety: Recombinant DNA, host cells, and process chemicals handled according to institutional guidelines

Standard Workflow

  1. Evaluate sequence, domain, signal peptide, tag, host, and vector
  2. Construct and sequence-verify expression clone
  3. Small-scale screen temperature, time, and induction or transfection conditions
  4. Scale up culture and harvest cells or culture supernatant
  5. Perform low-temperature lysis or clarify supernatant and control protein degradation
  6. Complete capture using affinity chromatography
  7. Add ion exchange or size exclusion polishing as required by purity and monomer needs
  8. Perform buffer exchange, concentration, sterile filtration, and aliquoting
  9. Complete purity, identity, aggregation, endotoxin, residual, and activity QC

Equipment, Reagents, and Consumables

  • Equipment: Shaker or cell culture system, refrigerated centrifuge, lysis equipment, chromatography system, ultrafiltration, electrophoresis, and quantification equipment
  • Reagents: Vectors and hosts, media and inducers, lysis and inhibitors, affinity resins, ion exchange or size exclusion packing materials, and storage buffers
  • Consumables: Culture vessels, filters, chromatography columns, ultrafiltration tubes, and low-binding storage tubes

Essential Controls and QC

  • Empty vector or uninduced control, soluble and insoluble fractions, input/flow-through/wash/elution fractions
  • SDS-PAGE or SEC purity, concentration and recovery, identity confirmation, and monomer ratio
  • Assess endotoxin, host residuals, sterility or bioburden, and functional activity based on intended use

Common Problems

Optimize vector, host, and induction when expression is low; lower temperature or change host when inclusion bodies are abundant; check tag exposure, pH, and ionic strength when resin binding is poor; optimize buffer and reduce concentration and freeze-thaw stress when aggregation occurs.

Product Selection Entry

Research Use Statement: Specific process parameters should be validated based on the target protein and final application.