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Analytical Chem JoVE (Open Access) Citable · DOI

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

DOI: 10.3791/59166-v
What you'll learn
  • ✓ Express eukaryotic borate transporters in S. cerevisiae using overexpression systems
  • ✓ Solubilize and purify membrane proteins from yeast membranes
  • ✓ Assess oligomeric assembly using glutaraldehyde chemical cross-linking
  • ✓ Adapt protocols for other challenging membrane protein targets
Protocol

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

Difficulty
advanced
Total time
~5–7 days (including yeast growth, expression, membrane prep, and purification)
Model organism
S. cerevisiae
Biosafety
BSL-1

Steps

1
Overexpress borate transporters in S. cerevisiae

Culture yeast cells and induce heterologous expression of borate transporter genes using standard overexpression vectors. Harvest cells at appropriate optical density to maximize protein yield.

▶ 00:28
2
Harvest and prepare yeast membranes

Lyse yeast cells via mechanical disruption or enzymatic digestion and isolate crude membrane fractions containing expressed borate transporters through differential centrifugation.

▶ 01:55
3
Solubilize and purify borate transporter protein

Treat membrane preparations with detergent to solubilize borate transporters, then perform affinity chromatography and size-exclusion chromatography to obtain pure, homogeneous protein.

▶ 03:51
4
Perform glutaraldehyde cross-linking assembly assay

Incubate purified borate transporter with glutaraldehyde to covalently cross-link multimeric complexes, then analyze by SDS-PAGE to determine oligomeric state.

▶ 05:56
5
Interpret purification and oligomerization results

Evaluate representative chromatograms, purity gels, and cross-linking patterns to confirm successful expression, solubilization, purification, and multimeric assembly of borate transporters.

▶ 07:09
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