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Over-expression of proteins using a modified pBAD24 vector in E. coli expression system.

Sampali Banerjee1, Shardul S Salunkhe, Anjali D Apte-Deshpande

  • 1Biotechnology R & D, Lupin Limited, Lupin Research Park, 46A/47A, Nande Village, Mulshi Taluka, Pune, 411042, India.

Biotechnology Letters
|March 31, 2009
PubMed
Summary

A modified pBAD24M vector significantly enhances prokaryotic protein expression. This improved system allows for efficient repression, modulation, and elevated expression of proteins like granulocyte colony stimulating factor and staphylokinase.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Protein Expression Systems

Background:

  • The pBAD24 vector is a commonly used prokaryotic expression system.
  • Achieving high-level expression of recombinant proteins can be challenging with standard vectors.

Purpose of the Study:

  • To engineer a modified pBAD24 vector (pBAD24M) with enhanced protein expression capabilities.
  • To evaluate the efficiency of the modified vector for expressing heterologous proteins.

Main Methods:

  • Construction of the pBAD24M vector incorporating the arabinose operon's araBAD promoter, T7g10 sequence elements, and a modified Shine-Dalgarno sequence.
  • Expression analysis of green fluorescent protein, granulocyte colony stimulating factor, and staphylokinase using both the original pBAD24 and modified pBAD24M vectors.

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  • Quantification of staphylokinase expression via ELISA.

    • Main Results:

    • The modified pBAD24M vector demonstrated significantly higher expression levels compared to the original pBAD24 vector.
    • Proteins like green fluorescent protein and granulocyte colony stimulating factor reached >35% of total cellular protein with pBAD24M.
    • Staphylokinase expression increased from 8 ng/10(6) c.f.u. with pBAD24 to nearly 55 ng/10(6) c.f.u. with pBAD24M.
    • Expression levels achieved with pBAD24M were comparable to those obtained with a T7 promoter-based system.

    Conclusions:

    • The modified pBAD24M vector is a simple and effective prokaryotic expression system.
    • pBAD24M facilitates efficient repression, modulation, and elevated protein expression.
    • This enhanced vector system offers a valuable tool for biotechnological applications requiring high yields of recombinant proteins.