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AthenaES™ Projects
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Expression of Human Lead-Binding Protein in E. coli and Pichia pastoris
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Client: In-house research project in collaboration with
Dr. Bruce Fowler, Director, Program in Toxicology, University of Maryland Medical
School.
Project Size: Phase I Small Business Innovative Research
Grant awarded by the National Institutes of Environmental Health Sciences,
National Institutes of Health
Duration: 9 months (not including current clinical studies)
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The purpose of this research was to develop an immunoassay to human
lead-binding protein (PbBP) and determine its clinical utility. Lead is a
pervasive pollutant. Because it is indestructible, much of the lead mined
over the last 5,000 years remains in the environment and its continued use
in manufactured goods makes it likely that human exposure will persist.
The detrimental health effects of lead exposure have been
well-documented. The lowering of the cut-off for medical intervention to
10 mg/dL, combined with data suggesting that cognition and neurobehavior
are adversely affected at these low exposures, makes it clear that methods
for assessing the consequences of low-level exposures is crucial for maintaining
human health standards. One means of measuring the effects of low-level
exposure is through biological markers. The aim of the research was to
assess PbBP as a biomarker for lead exposure. PbBPs are proteins with high
affinity and specificity for binding lead. In a rat model, PbBP was excreted
into the urine in response to exposure to low levels of lead in drinking water.
It follows that the human analogue may respond similarly and thus be a useful
biomarker for low-level lead exposure.
Native PbBP is difficult is obtain and purify in sufficient
quantities for generating antisera, therefore recombinant protein was used
to immunize animals for antisera production. To produce recombinant PbBP,
a synthetic gene encoding the entire PbBP was constructed. The synthetic
gene was made by PCR amplification of four oligonucleotides corresponding
to overlapping sections of the gene along with 5' and 3' primers. Briefly,
four 110 nucleotide long oligos, alternating sense/antisense and overlapping
by 20 bases, were annealed. This produced a 358 base gapped product. Upon
amplification in the presence of the 18 nucleotide-long primers, the gaps were
filled and the entire double stranded product amplified. To facilitate cloning,
restriction sites were incorporated into the 5' and 3' ends of the fragment.
The PCR product was ligated into plasmid pMALc2 (New England Bio Labs, Inc.,
Beverly, MA) and the ligation product used to transform E. coli strain CC118.
Ampicillin-resistant colonies were selected and purified, and strains harboring
plasmid DNA carrying the desired fragment were verified by DNA sequence. One
strain, designated WC1, was then used to express recombinant PbBP.
The expression strategy employed yielded PbBP fused to the maltose
binding protein (MalE) of E. coli. The MalE permitted purification of fusion
proteins by amylose affinity chromatography and aided in stabilizing the
expression. Between the carboxyl-terminus of MalE and the amino-terminus of
the desired protein was a Factor Xa cleavage site. Pilot experiments revealed
that the amylose affinity chromatography was not suitable for purifying the
MalE-PbBP product. This is likely due to disruption of the MalE function as
a consequence of the fusion. Therefore, an alternative approach for purification
was used.
E. coli strain WC1 was used to express PbBP. The purification
process was monitored by SDS-PAGE. Strain WC1 was grown in two 7.5-liter batch
fermentations with expression of MalE-PbBP induced by the addition of IPTG to a
final concentration of 1 mM. At 4 hours post-induction the cells were harvested,
extracts prepared by mechanical disruption using 0.1 mm glass beads and cellular
debris removed by centrifugation at 30,000 x g for 30 min. The fusion protein was
purified by ammonium sulfate precipitation: 50% saturation to remove the bulk of
the bacterial proteins, followed by 70% saturation to precipitate the fusion protein.
The protein was digested with Factor Xa at a ratio of 500:1, and the proteins
separated by preparative SDS-PAGE. Upon digestion of the fusion protein with Xa
protease, two polypeptides were produced: one of 45 kDa and a second of 11.7 kDa.
The predicted mass of the PbBP is 11.5 kDa, which corresponded well with the smaller
product. The larger polypeptide corresponded to the predicted size of 43 kDa for
the MalE expressed from pMALc2. The yield of recombinant PbBP was 1 mg per liter
of culture. The band corresponding to the rPbBP was excised from the gel and prepared
for injection into three rabbits.
The animals were immunized with 100 ?g of antigen in Freund?s complete
adjuvant initially and with 100 ?g antigen in Freund?s incomplete adjuvant on days
14, 21, 35, 49 and at three week intervals thereafter. Test bleeds were drawn on
day 28 and the first production bleed on day 60. Antisera production continued with
immunizations at 3-week intervals, with bleeds 10 days after injection. Antisera was
titered against pure rPbBP immobilized on polystyrene 96-well plates. The first
production bleeds had titers with dilution end-points of 1:20,000. No evidence of
reactivity to rPbBP was observed in the pre-bleed sera even at a dilution of 1:100.
The antibodies were purified by PbBP-affinity chromatography and used to develop an
immunoassay. The resulting ELISA is currently being used in studies to determine the
clinical efficacy of the test for monitoring exposure of children to lead.
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