Phuc H. B. Nguyen, Aaron Ong, Herbert Mao, Roger A. Acey

Department of Chemistry and Biochemistry, California State University 1250 N. Bellflower Blvd. Long Beach CA 90840 USA

Abstract:We have been developing a “heavy metal sponge” for the removal and/or recovery of toxic and

precious metal. The patented technology is based on a novel low molecular weight protein known

as metallothionein (MT). The protein has the unique ability to selectively bind a number of toxic and

precious metals. Here we show examples of MT’s metal binding activity with cadmium, europium,

lanthanide, and uranium. The metal binding activity of MT is unaffected in DMF and 30% acetone.

We have cloned the MT gene, expressed the protein in bacteria, and developed a simple, cost

effective purification scheme. Interestingly, expression of MT is pH dependent. We adsorbed MT to

a water permeable membrane that functions as a “metal sponge”. The MT removes metal from a

contaminated solution passing through the membrane toxic metal from a solution passing through

the membrane. The metal can also be removed by collecting MT metal complexes. MT was mixed

with seawater containing 100 ppt (trillion) of 109Cd and the complex collected using a membrane

centrifugal filter device. The flow through was assayed for radioactivity. The efficiency of metal

binding was determined by counting the radioactivity in the flow through. Under these conditions,

88% of the metal was removed from the seawater. Supported in part by a grant from CSUPERB.

.

Conclusions:

• MT has been successfully expressed and purified. Expression is pH dependent.

• MT retains its metal binding activity in 30%(v/v) acetone.

• Membrane bound MT retains its metal binding activity.

• MT is capable of removing metal at ppt (parts per trillion) from sea water

Results:

Acknowledgements: CSUPERB & CSULB Mini Grant Program

Introduction:Metallothionien (MT) is a low molecular weight, cysteine rich metal binding protein containing no

aromatic amino acids. MT has the unique ability to selectively bind toxic (or precious) heavy metals,

e.g., lead, mercury, and gold, in the presence of a large excess of sodium and calcium. The metal

binding activity is active under extremes of pH (4 to 10) and temperature (4 to100 oC) and occurs

instantaneously. The dissociation constants (Kd) for the metal-protein interaction are in the range of

10 -12 to10 -15. These properties make MT are well suited for environmental applications requiring

toxic or precious metal removal and/or recovery.

A “Heavy Metal Sponge” for Selective Removal and Recovery

of Toxic or Precious Metals from Water

Materials and Methods:

Expression and purification of MT protein

The MT gene was cloned into pET 11a. The construct is referred to as pCODAmt. Recombinant MT

was expressed in BL-21 E.coli . Medium at varying pH was inoculated with pCODAmt and incubated

overnight at 37 oC and 220 rpm. The following morning, a one liter LB culture was inoculated with 14

ml of the overnight culture. The cells were cultured to an A560 between 0.4-0.6. IPTG was added to

the culture to a final concentration of 1mM to induce expression of MT. Cells were collected and

lysed by sonication in 10mM Tris pH=8.0, 1ppm Zn2+, 0.1mM DTT, and 0.5mM PMSF. The lysate was

placed in boiling water for 10 minutes then clarified by centrifugation. The supernatant was spiked

with 1μl 109Cd and loaded onto a G-50 molecular exclusion column. The column was eluted with

50mM Tris, pH=8.0. Fractions containing MT were identified, pooled and concentrated using a

Milipore 3K MWCO membrane centrifugal device. The sample was loaded onto MonoQTM HR 5/5

anion exchange column and eluted with 300mM NaCl gradient in 20mM Tris, pH=8.0, 0.1mM DTT.

Peak fractions were pooled and concentrated using the described centrifugal device. The sample

was loaded onto a SuperdexTM HR 75/10 FPLC gel filtration column and eluted with 50mM PBS,

0.1mM DTT. Fractions containing MT activity were pooled and concentrated. Protein concentration

was determined and the purity of the preparation determined by SDS-PAGE 4-12% Bis-Tris gel.

Metal Sponge Proof - of - Principle

MT was adsorbed onto either Biodyne A or B. A solution of PBS containing 109Cd was passed through

the filter. The membrane was washed three time with PBS and assayed for radioactivity.

Metal Binding Activity

Five micrograms of metal was mixed with 400 ml of purified MT. The mixture was loaded onto a 5 ml

desalting column (ExcelluloseTM ) and eluted with PBS. Half-milliliter fractions were collected and

analyzed for metal by ICP-MS. For cadmium, 109Cd was added to a solution of MT to a final

concentration of 100 ppt (parts per trillion) and fractionated as described previously. Fractions were

analyzed for radioactivity. For metal binding in acetone, 200 ml of MT was mixed with 1 ml of 109 Cd,

loaded onto a 10 ml Sephadex LH-20 column, and eluted with acetone. Fractions were analyzed for

radioactivity

Metal Recovery from Sea Water

A solution of MT and 109Cd contaminated seawater was added to 1K MWCO membrane centrifugal

filter and centrifuged until the liquid had passed through the membrane. A solution of Tris buffer

served as control. Aliquots of the flow through and retentate were assayed for radioactivity.

Figure 1. Cadmium Binding Activity of MT. A solution of109Cd and MT was fractionated on a desalting column as described in Materials and Methods.

Figure 2: Lanthanide/Actinide Binding Activity

of MT. A solution of metal and MT was

fractionated on a desalting column as described

in Materials and Methods

Figure 5.: Removal of Toxic Metal from Seawater Using Centrifugal Units. 109Cd contaminated

sea water was mixed with a solution of MT. The mixture was concentrated using 1K MWCO

centrifugal device. The flow through was assayed for radioactivity.

Figure 6: Effect of pH on MT Expression. Cell

lysate obtained from each pH culture was

assayed for MT activity. Total protein was

also determined. The ratio between MT

activity and total protein for each pH was

graphed.

Figure 7: SDS-PAGE Analysis of Purified

MT. SuperdexTM fractions with significant

MT activity were pooled, concentrated

and loaded onto SDS-PAGE 4-20% Bis-Tris

gel.

References: Acey, R.A., Harpham, B., and Mustillo, M. Metal Binding Proteins and Associated Methods, US

Patent # 7,135,605. Issued 11/14/06.

Acey, R.A., Compositions and Methods for Removing Heavy Metals from Contaminated Samples

Using Membranes Provided with Purified Artemia Metallothionein(MT) Peptides, US Patent #

7,237,962. Issued 9/25/07

Figure 3: MT Metal Binding Activity in Acetone. Purified MT solution was spiked with 1μl 109Cd

and immediately fractionated on individual 5 ml Sephadex LH-20 column equilibrated with various

acetone concentrations. Fractions were counted for radioactivity.

MT MT MT

Toxic Metal

Sodium

Calcium

Membrane/Solid Support

Toxic Metal Free Solution

MT MT MT

Figure 4. Principle of Using Membrane Bound MT to Remove Toxic Metal from Water

.

Table 1: Proof-of-Principle Data. MT was adsorbed to the surface two different types of

Biodyne Nylon membranes. PBS containing 109Cd was passed through the membrane. The

membrane was then analyzed for bound radioactivity.

.

Membrane Only Membrane + MT Biodyne A 3768 152,876 Biodyne B 1774 158,762

4612 CPM 109Cd

585 CPM 109Cd