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Wheying in on protein production

Want to produce proteins in your lab on a budget? Tired of waiting for reagents to arrive? In this article, we share the experience of our partners at Open Bioeconomy Lab at the University of Cambridge with using lactose from milk, in place of isopropyl β-D-1-thiogalactopyranoside (IPTG), to induce protein expression in bacteria.

IPTG is a molecular biology reagent used for bacterial protein production. As a structural mimic of lactose, IPTG can bind and inactivate the lac repressor, triggering the transcription of genes under control of the T7 promoter. Although widely used, IPTG is expensive, costing around $19.5 per litre of media (final concentration 1 mM) in Cameroon.

Alternatively, purified lactose, or whey containing lactose, can be used to induce protein expression (1, 2). While purified lactose is difficult to find, whey is cheaply and easily obtained in Cameroon, costing around $0.08 per litre of media (final concentration 0.2%).

During their quest to express OpenVent (3), a highly thermostable DNA polymerase with five times higher fidelity than Taq, researchers in Open Bioeconomy Lab showed that both purified lactose and homemade whey could be used to induce OpenVent expression in a comparable manner to IPTG. Protocols and details of these experiments can be found in their open lab notebook on Benchling.

Comparison of expression of OpenVent DNA polymerase using IPTG, lactose and homemade whey as inducers
Comparison of expression of OpenVent DNA polymerase using IPTG, lactose and homemade whey as inducers. Bacterial cell lysates were run on an SDS-PAGE gel and visualised using Coomassie protein stain. Hours indicate the time elapsed since the culture was initiated. Asterisk indicates the position of OpenVent. Image provided by Open Bioeconomy Lab.

Although whey is cheap and easy to obtain, its preparation can be time-consuming. Considering this, the researchers tested whether untreated milk could produce similar results. When milk was added to the media it became cloudy, making measurements of bacterial density difficult. However, in terms of protein induction, the results were comparable to those seen with IPTG, purified lactose or whey.

Turbidity of media containing whey or boiled milk
Turbidity of media containing whey or boiled milk. Photograph was taken before inoculation with bacteria.

So where should you look to obtain lactose for your experiments? Fortunately, various milks and milk products contain high lactose content that could be exploited for bacterial protein expression, including cow milk, goat milk, condensed milk and whey, obtained as a waste product from the cheese industry (4).

Source

Lactose content (g/100 g)

Whole cow milk

3.7-5.1

Low-fat cow milk

3.7-5.5

Goat milk

4.1-4.7

Sweetened condensed milk

10.0-16.3

Cottage cheese whey

4.5-4.9

Condensed whey

38.5-39.0

Sweet dry whey

73.5-74.5

Lactose content of milk and milk products. Modified from “Chapter 3 Lactose content of milk and milk products”, 1988.

In conclusion, despite longer induction times, whey and milk offer attractive alternatives to IPTG for bacterial protein expression. With this in mind, it’s time to make your protein production legen-dairy!

If you are interested in protein expression, why not check out our bioreactor!

References

  1. Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005;41(1):207-34.
  2. Viitanen MI, Vasala A, Neubauer P, Alatossava T. Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli. Microb Cell Fact. 2003;2(1):2.
  3. Huang H, Keohavong P. Fidelity and predominant mutations produced by deep vent wild-type and exonuclease-deficient DNA polymerases during in vitro DNA amplification. DNA Cell Biol. 1996;15(7):589-94.
  4. Chapter 3 Lactose content of milk and milk products. The American Journal of Clinical Nutrition. 1988;48(4):1099-104.