Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni N utilization substance protein B homolog (nusB)

Basic Research Questions

• What expression systems are most effective for producing recombinant Leptospira interrogans nusB protein?

  Recombinant Leptospira proteins, including nusB, are most commonly expressed in Escherichia coli systems. For optimal expression of Leptospira interrogans nusB, the pRSET or pET expression systems with a His-tag are recommended. Expression in E. coli BL21(DE3) has shown good yields, typically 5-10 mg/L of culture when induced with 0.5-1.0 mM IPTG at OD600 of 0.6-0.8 .

  Alternative approaches include:

  • Expression as truncated regions if the full-length protein shows poor solubility

  • Fusion with solubility-enhancing partners such as GST or MBP

  • Codon optimization for E. coli to address the AT-rich bias in leptospiral genomes

  Expression System	Advantages	Disadvantages	Typical Yield
  pET with His-tag	High expression, simple purification	Potential inclusion bodies	5-10 mg/L
  GST fusion system	Enhanced solubility	Larger tag may affect function	3-8 mg/L
  MBP fusion system	Best solubility for difficult proteins	Large tag requires removal	4-7 mg/L

• What are the optimal conditions for purifying recombinant nusB protein from Leptospira interrogans?

  Purification of His-tagged nusB protein can be achieved through immobilized metal affinity chromatography (IMAC) using Ni-NTA resin. The optimal protocol includes:

  1. Cell lysis in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and 1 mM PMSF

  2. Binding to Ni-NTA resin in batch or column format

  3. Washing with increasing imidazole concentrations (20-50 mM)

  4. Elution with 250-300 mM imidazole

  5. Dialysis against storage buffer (typically PBS or Tris buffer with 6% trehalose)

  For higher purity, a secondary purification step using size exclusion chromatography is recommended. Typical purity after two-step purification exceeds 90% as determined by SDS-PAGE .

• How can I verify the identity and purity of recombinant nusB protein?

  Standard verification methods include:

  1. SDS-PAGE analysis to confirm molecular weight (expected ~20 kDa plus tag size)

  2. Western blotting using anti-His antibodies or specific anti-nusB antibodies

  3. Mass spectrometry for definitive identification and molecular weight confirmation

  4. N-terminal sequencing to confirm proper translation start

  5. Dynamic light scattering to assess protein homogeneity and aggregation state

  For functional validation, RNA-binding assays can be performed as nusB is involved in transcription regulation .

• What are the recommended storage conditions for recombinant Leptospira nusB protein?

  Based on storage practices for other Leptospira recombinant proteins:

  1. Store the purified protein in aliquots at -80°C for long-term storage

  2. Add 5-50% glycerol (final concentration) to prevent freeze-thaw damage

  3. Lyophilization in the presence of 6% trehalose can extend shelf life

  4. Avoid repeated freeze-thaw cycles as this significantly reduces activity

  5. Working aliquots can be stored at 4°C for up to one week

  The protein should be stored in a buffer containing Tris/PBS-based buffer at pH 8.0 with stabilizers such as trehalose .