PET28A SUMO plasmid 2 ug

Introduction

The pET28a-SUMO plasmid is a specialized derivative of the pET-28a(+) vector backbone that integrates a N-terminal 6×His-SUMO fusion tag upstream of the multiple cloning site (MCS). This design facilitates high-level expression, solubility enhancement, and site-specific proteolytic removal of fusion tags. The system is widely used for difficult-to-express or aggregation-prone proteins in Escherichia coli, particularly in structural biology, functional protein studies, and high-throughput protein screening.

Vector Backbone Architecture: pET-28a(+)

SUMO Tag Mechanism and Utility

SUMO (Small Ubiquitin-like Modifier) Tag

• ~11.2 kDa in size (101 amino acids from Saccharomyces cerevisiae SMT3)
• Enhances solubility, folding efficiency, and protease protection in bacterial cytoplasm
• Functions as a chaperone-like fusion partner
• Does not require additional linker or cleavage recognition sequence

Proteolytic Cleavage by SUMO Protease (Ulp1)

• SUMO-specific protease Ulp1 recognizes the tertiary structure of SUMO and cleaves exactly at the C-terminus of the tag
• Leaves no residual amino acids on the N-terminus of the target protein
• Cleavage occurs under mild, non-denaturing conditions (e.g., 20 mM Tris-HCl, 150 mM NaCl, pH 8.0)

Expression System Compatibility

• Optimized for BL21(DE3) and Rosetta(DE3) strains containing the T7 RNA polymerase gene under control of lacUV5
• Compatible with Lemo21(DE3) for tunable expression
• Expression is induced by IPTG at 0.1–1 mM final concentration
• Preferred expression temperature: 16–25°C to reduce misfolding and inclusion body formation

Protein Purification Workflow

1. Lysis: Lysozyme or sonication in native buffer, e.g., 20 mM Tris, 300 mM NaCl, 10 mM imidazole
2. Affinity Purification: Ni-NTA or cobalt-based IMAC via His₆-tag
3. Protease Cleavage: Addition of Ulp1 SUMO protease (typically 1:100 w/w enzyme:target) at 4–25°C for 2–16 hrs
4. Second IMAC: Flow-through contains tag-free protein (no His-tag), while tag and Ulp1 (His-tagged) bind to the column
5. Polishing Step: Gel filtration or ion exchange chromatography for final cleanup

Applications

Advantages over Other Tags

Limitations and Considerations

• Toxic or membrane-bound proteins may still require codon optimization or chaperone co-expression
• Codon bias in E. coli should be accounted for; use of Rosetta(DE3) strains recommended for AT-rich eukaryotic sequences
• SUMO-fused proteins may form oligomers depending on target folding properties
• Protein of interest must not contain internal SUMO cleavage sites unless verified

Protocol Optimization Tips

• Use 0.5 mM IPTG and express at 18°C overnight for maximum solubility
• Optimize lysis with DNase I + MgCl₂ to reduce viscosity
• Confirm cleavage via SDS-PAGE and western blot against SUMO or His-tag
• Remove Ulp1 by reverse IMAC or size exclusion chromatography if required