"Live" enzyme fusions

*.* Troianovsk_s at MSDISK.WUSTL.EDU
Wed Jan 25 22:06:06 EST 1995


>Date:   23-JAN-1995 05:20
>From: varyc.mmcri at OFFICE.MMC.ORG (Varyc)
>Description: "Live" enzyme fusions
>
>Hi All,
>Does anyone know if the GST enzymatic activity is expected to
>survive being fused to another polypeptide? <deleted>
> 
>Cal Vary

     Hi there, 
below some stuff about GST enzymatic (assay) activity. In my hands it worked
well when GST was fused with polypeptides ranged from 15-60 kDa. However,
see the Note.

   GST fusion proteins produced using a pGEX plasmid in E. coli may be
assayed using the GST substrate 1-chloro-2,4-dinitrobenzene (CDNB). This
assay can be used to optimize conditions for expression or to trace steps in
the purification of a GST fusion protein. GST from many sources, including
the schistosomal form used in the pGEX system, has a high affinity for CDNB
(1, 2). The enzyme catalyzes the conjugation of CDNB with glutathione and
results in a CDNB-glutathione product with a strong molar absorption at 340
nm ( = 9.6 mM-I cm-l). The reaction is relatively insensitive to reaction
conditions and, as such, the assay can be used with crude bacterial
sonicates or with purified GST fusion protein.

In this assay, a sample containing a GST fusion protein is incubated in a
reaction buffer containing CDNB and glutathione. The change in
absorbance at 340 nm is monitored for 5 minutes and based on these
results, the amount of GST fusion protein in the sample is calculated.
This assay can easily be used with multiple samples and is ideal for
screening E. C011 strains for expression, optimizing culture conditions for
expression, and for tracing the GST fusion protein during the purification
process.

- Note: The activity of the GST moiety can be affected by the folding
of the fusion partner. Thus, the absorbance readings obtained for a
given fusion protein may not reflect the actual amount of fusion
protein present.

REFERENCES

1. Habig, W. H. et al., J. Biol. Chem. 249, 7130 (1974).
9. Mannervik, B. and Danielson, U. H., CRC Crit. Rev. Biochem. 23,
283 (1988).
-----------
           Nikolai. 

Child says: "If All Fails Carefully Read the Manual"
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