Purification of the Highly Basic Proteins Avidin and Lysozyme by liaoqinmei

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									                                                                     Purification of the Highly Basic Proteins Avidin
                                                                     and Lysozyme Using Gradiflow Technology
                                                                                                      TM



                                                                     Rothemund D.L., Thomas T.M., Rylatt D.B.
                                                                     Gradipore Ltd, Sydney NSW 2086




 SUMMARY                                                              METHOD                                                          RESULTS

The basic proteins avidin pI 10.0 and lysozyme pI 10.7 have          Purification of Avidin and Lysozyme from Egg White             Purification of Avidin and Lysozyme from Egg White
been purified from chicken egg white using a charged based
strategy on the Gradiflow. The high pI of avidin and lysozyme                                                                                                                                                 1.     Start Sample Stream 2
allows them to be easily separated from the remaining egg                                                                                                                                                     2.     1 hour Stream 2
white proteins because at pH 9.0 avidin and lysozyme are the                                                                                                                                                  3.     2 hour Stream 2
only positively charged proteins. In a second step at pH 10.2                                                                                                                                                 4.     3 hour Stream 2
the positively charged avidin is separated from the negatively                                                                                                                                                5.     1 hour Stream 1
charged lysozyme. This procedure to purify avidin and                                                                                                                                                         6.     2 hour Stream 1
lysozyme took a total of 4.5 hours. Using the Gradiflow to                                                                                                                                                    7.     3 hour Stream 1
purify avidin and lysozyme is less complicated than other                                                                                                                                                     8.     Life Technologies
published methods and seemed to give higher recoveries                                                                                                                                                               Benchmark Prestained
without any requirement for column chromatography.                                                                                                                                                                   Protein Ladder
                                                                                                                                                                                                          Avidin
                                                                                                                                                                                                          Lysozyme

                                                                                                                                       1 2             3 4             5 6             7 8


                                                                                                                                    Purification of Avidin and Lysozyme
 INTRODUCTION                                                                        Charge Based Separation

                                                                                                                                                                                                              1.     Start Sample Stream 1
The Gradiflow is a preparative electrophoresis system for the        There were two charge based separations involved in the                                                                                  2.     30 minute Stream 1
purification of proteins. Separation of proteins using Gradiflow     purification of avidin and lysozyme from eggwhite. The first
                                                                                                                                                                                                              3.     60 minute Stream 1
technology is based on the charge and molecular weight of            charge based separation, at pH 9.0 took advantage of the
the protein. The charge of the protein can be varied                                                                                                                                                          4.     30 minute Stream 2
                                                                     high pI of avidin and lysozyme to separate them from the
depending on the pH of the buffer chosen. In the Gradiflow           remaining egg white proteins. A 1000kDa separation                                                                                       5.     60 minute Stream 2
the charged proteins can transfer through porous                     membrane allowed the positively charged avidin and                                                                                       6.     Life Technologies
polyacrylamide membranes towards either the cathode or the           lysozyme to rapidly move into Stream 1, away from the                                                                                           Benchmark Prestained
anode. The pore size of the membranes used in the Gradiflow          remaining negatively charged egg white proteins.                                                                                                Protein Ladder
                                                                                                                                                                                        Avidin
can range from 3kDa to 1000kDa and the choice of                                                                                                                                        Lysozyme
membrane size depends on the separation required.                                                                                    1        2 3 4 5                        6
                                                                     Purification of Avidin and Lysozyme
Egg white is a complex mixture that consists of more than 20         Avidin and lysozyme were then separated on the basis of
proteins. The two basic egg white proteins avidin and                their pI difference. At pH 10.2 avidin will be negatively
lysozyme constitute 0.05% and 3% of total egg white                  charged and lysozyme will be positively charged. A cartridge     DISCUSSION
proteins. Lysozyme has a molecular weight of 14kDa and has           with a 1000kDa separation membrane was utilised to allow
valuable anti-bacterial properties due to its ability to hydrolyse   the negatively charged avidin to move rapidly away from        Protein assays performed on the lysozyme fractions indicated
the ß-1,4-glycosidic linkages between N-acetylmuramic acid           lysozyme into Stream 2.                                        that 10-15mg of lysozyme with a total activity of 900000U -
(NAM) and N-acetylglucosamine (NAG) in bacterial cell                                                                               950000U was able to be purified from one egg white. Enzyme
walls.(1) Avidin has a molecular weight of 66kDa and is                                                                             immunoassay of avidin fractions obtained on the Gradiflow
composed of 4 identical subunits, each subunit is able to bind       Sample Analysis
                                                                                                                                    indicated recoveries of 60-65% from one egg white.
one molecule of biotin. The rapidly forming avidin biotin            Samples collected during the separations were analysed on
complex is one of the most stable non-covalent interactions          non-reduced 4-20% Tris-Glycine gels (Gradipore, Australia).    Using Gradiflow technology we were able to purify 950000U
known and has a dissociation constant of 10-15M.(2) Once             Recovery of biological activity of lysozyme was assessed by    of lysozyme and 2.5mg of avidin from one egg white.
formed, the avidin biotin complex is resistant to a wide range       the rate of lysis of Micrococcus leutus and the recovery of    Gradiflow purification of avidin and lysozyme from egg white
of pH values, temperatures and organic solvents.(3) The              avidin was assessed by an enzyme linked immunosorbent          is less labour intensive and results in better yield than other
avidin-biotin complex is widely used as a detection reagent          assay (ELISA). The protein content of the lysozyme fractions   published methods including ion exchange or affinity
for antibodies and proteins.                                         was determined by BCA reagent (Pierce, Rockford, IL, USA).     chromatography on iminobiotinyl-Sepharose.


Figure 1. Gradiflow Instrument and Cartridge Assembly
                                                                                                                                      REFERENCES
                                                                                                                                    1. Holler, E., Rupley, J., and Hess, G. (1974). FEBBS Lett. 40, 25-
                                                                                                                                    2. Green, N. M. (1963). Biochem. J. 89, 585-591
                                                                                                                                    3. Green, N. M. (1975). Adv. Protein Chemistry. 29, 85-133



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