Introduction


History


Gene and Protein Isolation


Structure and Mechanism


Biological Significance


Summary


References





BCMB 8010 website

Please note these web pages are part of an assignment for a graduate course in Advanced Biochemistry and Molecular Biology BCMB8010 at the University of Georgia. Questions should be directed to Gina Pries (gpries@uga.edu).

EPSP Synthase


Gene and Protein Isolation

AroA isolation and cloning
The AroA gene, coding for the E. coli EPSP synthase, was first isolated from a lambda transducing phage (lambda-serC) found to contain a portion of the E. coli chromosome (16). The location of AroA (then designated S1) had also been mapped to its region on the E. coli chromosome (17). AroA was subcloned by restriction cuts from lambda-serC (18), but it was not until 1984 that AroA was sequenced (19), thereby allowing much more efficient plasmid construction without extra chromosomal DNA on either side of AroA. With the sequence revealed, plasmid construction continually improved to yield greater amounts of EPSP synthase produced in E. coli which also yielded more efficient purification techniques.

EPSP synthase purification
EPSP synthase was first purified as described by Lewendon and Coggins (20), but the method has since been improved through the use of a better vector employing the T7 promotor in E. coli (J). With this new expression system, EPSP sythase comprises approximately 50% (Table 1) of the total cell protein as compared to 2% with a previous expression system (21). The system described by Shuttleworth, et al. (21), involves transformation of the E. coli strain BL21 (22) with an AroA-containing vector utilizing the T7 gene 10 promotor which is strongly expressed when induced with isopropyl-1-thio-ß-D-galactopyranoside (IPTG) in BL21 cells (21).

Table 1 Purification stages and yield for EPSP synthase adapted from Shuttleworth et al. (21)
StepTotal protein (mg)Activity (units)Specific activity (units/mg)Yield (%)Purification factor
Crude extract86459686.91001
50-70% Ammonium sulfate703----
Phenyl-Sepharose360483713.4811.9
Mono-Q260384014.8642.2

The purification strategy of Shuttleworth et al. involves a minor modification of other strategies (18, 23) and is similar to the first purification strategy described by Lewendon and Coggins (20). After cell lysis, the crude extract is saturated to 50% with ammonium sulfate and centrifuged, and the resulting supernatant is saturated to 70% with ammonium sulfate and centrifuged a second time (21). The pellet is collected, buffered, and loaded onto a pheyl Sepharose column which is then eluted with decreasing concentrations of ammonium sulfate (21). Enzyme-containing fractions (assayed for activity) are desalted and loaded onto a PD10 column after which the eluant is subjected to FPLC™ on MonoQ 16/10 with an increasing KCl gradient (21). Throughout the strategy, it is important to add ß-mercaptoethanl or DTT to buffers to eliminate the multiple oxidized forms of the enzyme (21).

Activity assays
When EPSP synthase was first isolated, two assay methods were employed to detect activity. Both couple the EPSP synthase reaction with other enzymes which produced detectable products. In the forward direction, EPSP synthase can be coupled with chorismate synthase, the enzyme in the Shikimate pathway which converts EPSP to chorismate; as EPSP synthase produces EPSP, chorsimate synthase can convert EPSP to chorismate which can be detected at 275 nm (20). Since EPSP synthase can also proceed in the reverse direction, activity can also be assayed with coupling to pyruvate kinase and lactate dehydrogenase which oxidize NADH in the breakdown of pyruvate, allowing the detection of NADH loss at 340 nm which corresponds to pyruvate evolution by EPSP synthase (20). This latter method is the most commonly used to assay EPSP synthase activity currently.





Updated on 11/13/02
Author: Gina Pries (gpries@uga.edu)