Augmedium Case Studies Technical Brief
Sheldon E. Broedel, Jr., Ph. D.
Athena Environmental Sciences, Inc., Baltimore , MD
Introduction
Augmedium™ is a medium additive used to precondition cultures for
improving the expression of recombinant proteins in E. coli which
tend to form insoluble products, inclusion bodies and aggregates. Frequently,
heterologous proteins, when highly expressed in E. coli, accumulate as
insoluble products. The protein produced under these circumstances is most
often inactive, and, furthermore, it can be difficult or impossible to recover
functional protein from the insoluble material. While techniques are available
for purifying and refolding proteins which are produced as inclusion bodies
this is not always desirable.
The role of molecular chaperones in protein folding has been extensively
studied, 1,2,3. In E. coli the two primary chaperone
networks are DnaK-DnaJ-GrpE and GroEL-GroES. In addition to these two networks there
are several minor chaperones the expression of which are induced when the cells are
under heat, chemical and oxidative stress. The chaperone proteins have been proposed
to interact with nascent polypeptides and to facilitate the correct folding. Thus,
it is not unexpected that when DnaK-DnaJ-GrpE or GroEL-GroES complexes are overexpressed
the solubility of a number of aggregation-prone proteins is improved. 4,5,6,
7,8,9,10,11,12,13,14. However, not all insoluble proteins
exhibit improved solubility with overexpression of DnaK-DnaJ-GrpE or GroEL-GroES. Moreover,
it has been shown that the solubility of some proteins is increased when the cells are
subjected to chemical, thermal and oxidative stresses before expression of the insoluble
protein. 15,16,17,18. Therefore, it seems likely that other chaperones
may be necessary for some proteins. However, the mechanism by which a given protein is
recognized by any given chaperone protein is not known. Augmedium™ was thus designed
to induce the expression of several different chaperone proteins thereby allowing for
an improvement in the solubility of aggregate-prone proteins without the need for
identifying a specific chaperone effector. Below are two case studies where Augmedium™
was used.
The first case was an esterase from Vibrio cholera. This protein
was expressed using pQE31 (Qiagen) with an N-terminal His tag in the strain M15. The
protein accumulated to a large extent as an inclusion body with little of the protein
accumulated in a soluble form. To increase the recovery of soluble enzyme, we first
examined the effect of culture medium. A medium screen was performed according to
the protocol of the Medium Optimization Kit™ (AthenaES™). Soluble protein
was determined by measuring the level of enzymatic activity present in cells
extracted with Y-Per Buffer (Pierce Chemical). It was found that the amount of
enzyme activity recovered was medium-dependent and that Hyper Broth™ yielded
the highest level of enzyme activity (Fig. 1). This was in contrast to LB (Miller)
Broth where no enzymatic activity was detected.
Figure 1. Medium-dependent accumulation of LypA after induction of expression.
To determine whether Augmedium™ could improve the recovery of a protein
in a medium giving poor expression, expression of LypA was induced in cells grown in
Power Broth. This medium gave a low but measurable level of activity (Fig. 1). The
effect of Augmedium™ on LypA activity was examined by culturing the cells in 25ml
of medium to a density of 1.0 OD600 and adding Augmedium™ to the culture
at five different concentrations 20 min. prior to adding IPTG to 1mM. After 3 hours
incubation, the cells were harvested and the soluble enzyme released using 1ml Y-Per
Buffer (Pierce Chemical). LypA activity was measured and the specific activity determined.
A dose-dependent increase in enzyme activity with increasing Augmedium™ concentration
was observed (Fig. 2). The Augmedium™ at a concentration of 2.5x increased the
yield of soluble esterase 5-fold over the non-treated culture.
Figure 2. Augmedium™-dependent increase in LypA activity. IGP isogenic parent
showing endogenous esterase activity. Samples were 3h post-induction.
In another example, AES8 (the functional properties of the protein can not be
disclosed at this time due to its proprietary status), a somewhat more complex expression
pattern was observed. As above, a screen of six medium formulations (Medium Optimization
Kit™, (AthenaES™) was used to determine the one yielding the highest level of
soluble protein accumulation. Maximum levels of active protein in the soluble fraction
were found when the cells were cultured in Glucose M9Y™ though the fraction of
soluble AES8 protein produced remained less than 10% of the total accumulated. To increase
the amount of soluble protein, the Augmedium™ concentration was titered in a matrix
experiment (fractional factorial design) along with different IPTG concentrations and
induction times. For this protein, both an enzyme assay and immunoassay were used to determine
the level of soluble protein.
With regard to enzyme activity, a time- and Augmedium™ dose-dependent
(pre-condition) increase in protein accumulation was found (Fig. 3). Maximum activity was
achieved after 6 h induction with 0.53 mM IPTG and 1x Augmedium™. With respect to
AES8 mass accumulation (as measured by immunoblot), there appeared to be an interaction
between the IPTG and Augmedium™ with maximum accumulation at the extremes of the
dosing range and minimum in the mid-range doses (Fig 4.). These findings suggests that
some portion of the protein that accumulates is not active. Therefore, when interpreting
data on the production of a given recombinant protein caution is advised against basing
conclusions solely on mass accumulation data.
Figure 3. The increase in AES8 activity as a function of Augmedium™
concentration and induction time.
Figure 4. Accumulation of AES8 as a function of Augmedium™ and IPTG concentrations.
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