Product Overview

The pET-28a vector is one of the most widely used E. coli protein expression plasmids in molecular biology and structural biology research. Originally developed by Novagen (EMD Biosciences / Merck), the pET system has become the gold standard for high-level, IPTG-inducible recombinant protein expression in bacterial hosts.

pET-28a combines the powerful T7/lac hybrid promoter system with an N-terminal His-Tag, T7-Tag, and thrombin cleavage site, plus an optional C-terminal His-Tag for dual-tag purification strategies. This makes it exceptionally versatile — allowing researchers to express and purify target proteins via Ni-NTA affinity chromatography, and then remove the N-terminal fusion tags with thrombin protease if desired.

With a high copy number (approximately 40–60 copies per cell, based on the pBR322 origin of replication), pET-28a ensures robust plasmid yield during preparation and supports high-level target gene expression upon IPTG induction in DE3 lysogen strains such as BL21(DE3).

Key Features

Technical Specifications

Property	Value
Plasmid Type	E. coli Expression Vector
Vector Size	5369 bp
Copy Number	High (pBR322 origin, ~40–60 copies per cell)
Promoter	T7/lac hybrid promoter (IPTG-inducible)
Antibiotic Resistance	Kanamycin (Kanr), 30–50 µg/mL working concentration
N-terminal Tags	6×His-Tag, T7-Tag (MASMTGGQQMG), Thrombin Cleavage Site
C-terminal Tag	6×His-Tag (optional, removable by introducing stop codon)
Cloning Method	Restriction enzyme digestion & ligation via MCS
5' Sequencing Primer	T7 Promoter: 5'-TAATACGACTCACTATAGGG-3'
3' Sequencing Primer	T7 Terminator: 5'-GCTAGTTATTGCTCAGCGG-3'
Recommended Host Strain	BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), Origami(DE3)
Storage	-20°C (short-term); -80°C (long-term)
Biosafety Level	BSL-1

Multiple Cloning Site (MCS)

The pET-28a MCS contains unique restriction enzyme recognition sites arranged sequentially downstream of the N-terminal fusion tags and thrombin cleavage site. The following restriction enzymes are available for cloning:

Note: pET-28a differs from pET-28b(+) and pET-28c(+) only in the translational reading frame of the MCS relative to the N-terminal tags. Select the variant that places your target gene in the correct reading frame.

Application Guide & Protocols

1. Recommended Expression Workflow

1. Clone Target Gene into MCS: Digest pET-28a and insert DNA with compatible restriction enzymes. Ligate and transform into a standard cloning strain (e.g., DH5α). Verify the construct by colony PCR, restriction digestion, and Sanger sequencing using T7 Promoter and T7 Terminator primers.
2. Transform into Expression Host: Transform the verified plasmid into BL21(DE3) or a compatible DE3 lysogen strain. Plate on LB agar containing 50 µg/mL kanamycin and incubate at 37°C overnight.
3. Small-Scale Expression Screening: Pick a single colony and inoculate 3–5 mL LB + Kan (50 µg/mL). Grow at 37°C, 220 rpm until OD₆₀₀ = 0.6–0.8. Induce with 0.1–1.0 mM IPTG. Test expression at various temperatures (16°C, 25°C, 30°C, 37°C) and durations (2–20 hours).
4. Scale-Up & Harvest: Scale the optimized conditions to desired culture volume. Harvest cells by centrifugation at 4000 × g, 4°C, 15 min. Cell pellets may be stored at -80°C or processed immediately.
5. Ni-NTA Affinity Purification: Resuspend pellet in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF). Lyse by sonication or French press. Clarify at 12000 × g, 4°C, 30 min. Apply supernatant to Ni-NTA resin. Wash with 20–50 mM imidazole. Elute with 250–500 mM imidazole.
6. Optional Tag Removal: Dialyze or buffer-exchange purified protein into thrombin cleavage buffer (20 mM Tris-HCl pH 8.4, 150 mM NaCl, 2.5 mM CaCl₂). Add thrombin protease (1 U per mg of target protein). Incubate at room temperature for 2–16 hours. Remove cleaved tags and thrombin by re-passing through Ni-NTA and/or benzamidine resin.

2. IPTG Induction Optimization

Parameter	Recommended Range	Starting Recommendation
IPTG Concentration	0.01–1.0 mM	0.5 mM
Induction OD600	0.4–1.0	0.6–0.8
Post-Induction Temperature	16–37°C	30°C (soluble proteins) 37°C (insoluble/inclusion bodies)
Post-Induction Duration	2–20 hours	4 hours (37°C) 16 hours (16–25°C)
Kanamycin Concentration	30–50 µg/mL	50 µg/mL

3. Troubleshooting Common Issues

Issue	Possible Cause	Solution
No / low protein expression	Incorrect reading frame or promoter incompatibility	Verify construct by sequencing; confirm use of DE3 lysogen host
Protein in inclusion bodies	Expression rate too high; incorrect folding	Lower IPTG to 0.1 mM; reduce induction temperature to 18–25°C; use BL21(DE3)pLysS
Leaky expression (toxicity)	Basal transcription from T7 promoter	Add 1% glucose to culture medium; use BL21(DE3)pLysS; use pLysS or pLysE strains
Poor Ni-NTA binding	His-Tag buried or degraded	Add 10–20 mM imidazole to lysis buffer; include protease inhibitors; try C-terminal His-Tag
Low plasmid yield	Degraded or contaminated preparation	Use fresh transformant; verify Kan selection; use endA- host for plasmid prep

Comparison: pET-28a vs pET-28b(+) vs pET-28c(+)

The three pET-28 variants — a(+), b(+), and c(+) — are identical in backbone sequence, antibiotic resistance, promoter system, and fusion tags. The only difference is the translational reading frame of the multiple cloning site (MCS) relative to the N-terminal His-Tag / T7-Tag / thrombin site fusion. This creates a set of three vectors that allow cloning into all three possible reading frames, ensuring that at least one vector will produce an in-frame fusion with the N-terminal tags regardless of the restriction sites used.

Frequently Asked Questions

Related Products

Recommended Expression Host Strains

Related pET Vectors

Accessories & Reagents

Alternative Names & Synonyms

pET28a, pET-28a, pET28a(+), pET-28a vector, pET28a plasmid, pET-28a expression vector, Novagen pET-28a, His-Tag expression vector, T7 promoter plasmid, Kan^r expression vector