Ali Wright and Natalina Elliott

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Verifying the Recommended Optimal Density for
Protein Expression in E. coli BL21(DE3)pLysE

• Ali Wright and Natalina Elliott

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Introduction

• Recombinant proteins are becoming of more value
  in biotechnology. Many of them, such as insulin,
  are of medical value. Plasmids, such as
  pcDNA3.1/His B/lacZ SD, are used to produce large
  amounts of these proteins. However, the amount
  of protein is determined by the conditions in
  which the bacteria harboring the plasmid exist.
  When bacteria are induced to produce large
  amounts of recombinant protein they devote the
  majority of their energy solely to making that
  protein. The more energy the bacteria has to
  make the protein, the more protein is produced.
  The bacterias energy is limited by the resources
  available to it, making the density of the
  culture an important concern. In dense cultures
  the bacteria have few resources, therefore they
  have less energy to devote to making the desired
  protein. If the bacteria are induced when the
  culture is less dense (and therefore has more
  resources available to the bacteria) then more
  protein will be produced. It has been reported
  that the O.D. for induction to produce the most
  protein was 0.4. The aim of this experiment was
  to test that observation by quantifying the
  amount of protein produced at varying O.D.s.

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Methods

• pcDNA3.1/His B/lacZ SD was transformed into E.
  coli BL21(DE3)pLysE.
• A single colony was grown in LB broth using
  ampicillin to maintain the plasmid and
  chloroamphenical to select for the bacteria.
• 150 dilutions of the culture were analyzed at
  O.D.s of approximately 0.2, 0.4, 0.8, 1.0, and
  1.2.
• Each sample was induced with IPTG for two hours
  once the desired O.D. was achieved.
• All protein was isolated and quantified using a
  NanoOrange Kit.
• The His6 tagged ?-galactosidase protein was
  isolated using Ni-NTA agarose.
• A second assay was performed to quanitify the
  purified protein (see Figure 1).

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Purified Protein Assay
Figure 1. Protein samples were assayed using the
NanoOrange Kit. The top row is the BSA standard
and the bottom two rows are undiluted purified
protein and a 110 dilution of the purified
protein.
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Figure 2. BSA was used as a control against
which we measured the purified protein from the
samples.
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Figure 3. These points were plotted based on the
absorbance values which were plugged into the
equation from figure 2. The resulting
concentrations were then plotted against the O.D.
for each sample.
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Results

• The undiluted sample with the O.D. value of
  approximately 1.0 appeared to have the highest
  concentration of ?-galactosidase (see Figure 3).
• However for the diluted samples the concentration
  of ?-galactosidase peaked at about and O.D. of
  0.4.
• These concentrations were determined by
  comparison to the control, BSA (see Figure 2)

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Discussion

• The results from this experiment are
  inconclusive due to discrepancies between the
  undiluted and the 110 diluted assay samples (see
  Figure 3).
• These discrepancies could be offset by
  increasing the sample size.
• It is currently recommended that the O.D. for
  induction is 0.4.

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References Acknowledgments

• Doyle, Ken. Protocols and Applications Guide
  Third Edition. USA Promega Corporation. 1996.
• Dr. Rinehart Verbal Instruction Spring 2004.
• Scope, R. K., Protein Purification, Principles
  and Practices, 2nd edition, Springer-Verlag
  (1987) NanoOrange Protein Purification Kit
  (N-6666)(2001).
• QIAexpressionist A Handbook for high-level
  expression and purification of 6xHis-tagged
  proteins, 5ht edition June 2003.

• Dr. Claire Rinehart
• Western Kentucky Biology Department
• Biotechnology Center
• Biol 450 Class of Spring 2004