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PrIME Purification of
Recombinant Hirudin
Contributed by: Pier Giorgio Righetti1, Alessandra Bossi1, Carlo Visco2,
Umberto Breme2, Maurizio Mauriello2, Barbara Valsasina2, Gaetano
Orsini2 and Elisabeth Wenisch3
University of Milano, L.I.T.A., Via Fratelli Cervi 93, Segrate 20090, Milano
1
Amersham Biosciences via Giovanni XXIII, I-20014 Nerviano (Italy)
Application Note #14
IsoPrime®
15
1
Val Ser Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Tyr Cys Leu
2
University of Forestry and Agriculture, Vienna, Austria
3
Keywords: PrIME, IsoPrime, Hirudin, Preparative Isoelectric Focusing,
Protein Purification
Introduction
Hirudin is a potent and selective inhibitor of thrombin, a
serine protease required for blood clotting. More than twenty
highly similar isoforms of hirudin have been isolated from
leeches belonging to the Hirudinidae family [1]. Because of its
therapeutic potential, we studied the problem of protein
micro-heterogeneity in a novel recombinant hirudin variant,
HM2, from the Asian leech Hirudinaria manillensis [2],
produced in Escherichia coli.
Therapeutic application of recombinant proteins requires
extensive characterization of purity and identification of
protein micro-heterogeneities which can occur during biosynthesis, within the producing cells, or during the downstream
purification process [3]. Here we describe the use of analytical and preparative immobilized pH gradient and pH
membrane techniques in the identification and isolation of a
number of minor degradation derivatives of HM2 for further
characterization by HPLC, mass spectrometry, sequence
analysis, and peptide mapping.
Results
Recombinant hirudin variant HM2 was expressed in E. coli
as an OmpA leader peptide-HM2 fusion protein. The
primary translation product was exported into the bacterial
periplasmic compartment and processed by endogenous
leader peptidase I to release the mature form of HM2—64
amino acid residues with three disulfide bonds (Figure 1).
A simple purification protocol with two column chromatography steps gave a preparation of HM2 more than 95%
pure, as demonstrated by RP-HPLC [2]. Analytical IPG-IEF
on slab gels (Figure 2 B, lane 1), revealed the major band of
HM2 at pI 4.03 and four minor bands, three more basic (pI
80-6383-24
Rev A / 6-97
e
30
16
Cys Val Gly Ser Asn Val Cys Gly Glu Gly Lys Asn Cys Gln Leu
a
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31
Ser Ser Ser Gly Asn Gln Cys Val His Gly Glu Gly Thr Pro Lys
c
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46
Pro Lys Ser Gln Thr Glu Gly Asp Phe Glu Glu Ile Pro Asp Glu
61
64
Asp Ile Leu Asn
b
Figure 1. Primary sequence of HM2. Recombinant hirudin variant
HM2 is a polypeptide of 64 amino acids with disulfide bonds between
Cys6-Cys14, Cys16-Cys28 and Cys22-Cys37. The positions of proteolytic
cleavages are indicated with arrows.
of 4.10, 4.25 and 4.31) and one more acidic (pI 3.98). PrIME
(preparative isoelectric membrane electrophoresis) separation
of HM2 components was carried out in a multi-compartment
electrolyzer ( IsoPrime) set up with pI-selective
membranes having defined pHs of 3.0, 4.0, 4.19 and 5.0.
The two more basic forms were isolated in a single PrIME
fraction (Figure 2 B, lane 4), and then separated further by
RP-HPLC. The two contaminants remaining with the correct
HM2 product were separated by micro-preparative IPG-IEF
on slab gels with an immobilized pH gradient from pH 3.5 to
pH 4.3 (Figure 2 A). The four isolated contaminant forms
were analyzed by a combination of RP-HPLC, mass spectrometry, peptide mapping and limited proteolysis
experiments.
The major contaminant was the more acidic component e
(pI 3.98), approximately 3% of the main protein HM2, the
other three impurities were each ² 0.5% (Figure 3, upper
panel). N-terminal sequence analysis of component e
produced the sequence: Tyr3-Thr-Asp-, indicating the loss of
the first two residues from the HM2 molecule. Mass spectrometry of component e gave a molecular mass of 6610 Da,
matching the calculated value for a protein consisting of
residues 3 through 64 of HM2.
N-terminal sequence analysis of component a (pI 4.31)
produced the sequence Val17-Gly-Ser-Asn-Val-, Val being at
position 17 of the HM2 chain. Cleavage at position 47 with
trypsin followed by RP-HPLC separation of the two resulting
peptides yielded a fragment whose sequence was found to
match the C-terminal sequence from positions 48 to 64.
Finally, mass measurements of component a gave a mass of
5032 Da, corresponding to the calculated mass of the peptide
chain 17-64 of HM2.
Component b (pI 4.25) had the correct N-terminal sequence
of HM2: Val1-Ser-Tyr-Thr-Asp-; however, the mass was
6212 Da, 585 Da less than full length HM2, suggesting the
loss of the last five carboxy-terminal residues. Selective
trypsin cleavage at position 47 followed by complete
sequencing of the resulting C-terminal fragment gave the
sequence from residue 48 to 59, confirming that component
b corresponded to the peptide chain 1-59 of HM2 (Figure 4).
The N-terminal sequence of component c (pI 4.10) was Val38His-Gly-Gln-, Val being at position 38 of the HM2 chain.
ES-MS analysis gave a mass value of 2980 Da, corresponding
to the peptide chain 38-64 of HM2.
Figure 2. Isoelectric focusing of recombinant hirudin variant HM2 on
immobilized pH gradients. Panel B: Analytical separation of HM2
(pI 4.03) in the pI range 3.60-4.50 showed four minor components
(lane 1). Separation in the IsoPrime unit (lanes 2, 3 and 4, corresponding
to chambers 1, 2 and 3 respectively) yielded the two more basic species a
(pI 4.31) and b (pI 4.25) in one chamber, well resolved from the principal full length form. Panel A: A micro-preparative slab gel, spanning the
pH range of 3.50-4.30, was used to separate the other two minor species
c (pI 4.10) and e (pI 3.98).
Discussion
Heterologous recombinant proteins produced in E. coli can
undergo intracellular proteolysis by action of cytoplasmic
proteinases. Mass spectrometry has recently proven to be an
important methodology for characterizing peptides and
proteins, particularly when combined with other techniques,
such as gel electrophoresis and amino acid sequence analysis
[4]. The coupling of SDS-denaturing gel separation of
proteins with matrix-assisted laser-desorption time-of-flight
mass spectrometry (MALDI-TOF-MS) techniques has been
described for proteins blotted onto a membrane [5, 6], and
proteins extracted from agarose gel slices [7]. However, in
these applications, essentially the same information, the
molecular mass of the protein, was provided by two different
methods. The approach described here, IPG followed by MS,
characterizes protein variants by both isoelectric point (pI)
and molecular mass, a true 2-D technique, giving the most
accurate values for pI and mass.
3 examples of this phenomenon. Full length hirudin has a pI
of 4.03:
Component a (pI 4.31, amino acids 17-64) lost a stretch
of 16 amino acids from the N-terminus containing 2 acidic
residues (Asp and Glu). Its pI increased to 4.31, a DpI of
+0.14/acidic residue;
●
Component b (pI 4.25, amino acids 1-59) lost a
pentapeptide at the C-terminus containing 2 acidic residues
(Asp and Glu). Its pI increased to 4.25, a DpI of
+0.11/acidic residue; and
●
Component c (pI 4.10, amino acids 38-64) lost a stretch
of 37 amino acids from the N-terminus containing 1 Asp,
2 Glu and 1 Lys. Here the DpI is rather minute: +0.07
because, at pH = pI, it takes more than two Glu residues to
neutralize one Lys since the Glu is only partially ionized.
●
The present data highlight one important mechanism of postsynthetic protein modification leading to a higher pI form.
While a variety of modifications leading to lower pI species
are well documented in the literature, no mechanisms leading
to generation of higher pI forms from a parent macromolecule have been advanced so far. It is now evident from our
data that at least one clear mechanism can be identified:
proteolytic cleavage. The hirudin contaminants presented
Because component e (pI 3.98, amino acids 3-64) lost only a
neutral dipeptide at the N-terminus (Val-Ser), the small negative DpI must be ascribed to minute pK variations of some
charged residues in the native structure.
2
Conclusion
d
100
Absorbance at 215 nm
The use of preparative and micro-preparative isoelectric
focusing techniques using pI-selective membranes (PrIME)
and immobilized pH gradient gels for protein purification in
combination with conventional chromatography, mass spectrometry and amino acid sequencing is a powerful means of
elucidating the nature of protein modifications. This knowledge is very valuable in the genetic engineering of host strains
and the design of larger scale production and purification
processes.
80
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b
c
e
a
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0
10
20
30
40
50
60
Time (min)
Materials and methods
100
Absorbance at 215 nm
Reversed-phase high pressure liquid
chromatography (RP-HPLC)
RP-HPLC was performed on a Hewlett-Packard 1090M
liquid chromatographic system with a Vydac 218TP C18
column, 4.6 x 250 mm, (The Separation Group, Hesperia,
CA, USA). Mobile phases A and B were H2O and acetonitrile
containing, respectively, 0.1 and 0.078 % trifluoroacetic acid.
A linear gradient from 18 to 26% of phase B was applied
over 46 min at a flow rate of 0.45 ml/min. The elution profile
was monitored with a Hewlett-Packard 1040A diode array
detector at 215 nm.
a
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e
b
c
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0
10
20
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60
Time (min)
Electro-spray mass spectrometry
Figure 3. Analytical RP-HPLC of hirudin variant HM2 and its degraded derivatives. Elution profile of HM2 preparation (upper panel) showed
the main peak of HM2 (peak d, approximately 95% of total peak area)
and its degraded components (peaks b, c, e and a). The degradation
products, after separation by IEF on IPGs, were analyzed by RP-HPLC
as shown in the lower panel.
Molecular mass measurements were performed on a HP
5989 A MS-Engine single quadrupole instrument equipped
with a HP 59987 A electro-spray interface (Hewlett Packard,
Wilmington, DE, USA). Samples recovered from the IsoPrime
multi-compartment electrolyzer or from micro-preparative
IPG gels were diluted with 50% methyl alcohol-H2O
containing 1% acetic acid and injected into the ion source at
a flow rate of 2-5 µl/min. The electro-spray potential was
approximately 6 kV; the quadrupole mass analyzer was set to
scan over mass-to-charge ratios (m/z) from 1000 to 1700 at
2 s per scan for a total time of 10-12 s. The sum of data
acquired over this time constituted the final spectrum.
Molecular masses were calculated from several multiply
charged ions within a coherent series. Calibrations were
performed with horse skeletal muscle myoglobin.
Standard manufacturer’s procedures and programs were used
with minor modifications.
Immobilized pH gradients (IPG)
IEF in IPG was performed on an Amersham Biosciences
Multiphor® II flatbed electrophoresis unit. Gels contained
5%T, 4%C and spanned the intervals pH 3.6-4.5 or pH
3.5-4.3. The formulations for these IPG ranges were from
Righetti [8]. After polymerization, the gels were washed,
dried and re-swollen in 20% glycerol. Gels were run for 2 h
at 400 V, then 12 h at 2000 V, 10 °C. Since hirudin is very
poorly stained with Coomassie Blue R-250, at the end of the
run the gels were blotted onto PVDF membranes and stained
with Ponceau S. Hirudin and its minor isoforms appeared as
intense red/pinkish zones on a white background.
Sequence analysis
N-terminal sequence analysis was performed by automated
Edman degradation using a pulsed liquid-phase sequencer
model 477 A with an on-line analyzer model 120 A (PerkinElmer/Applied Biosystems, Foster City, CA, USA ) for the
detection of phenylthiohydantoin derivatives of amino acids.
3
Preparative purification in the IsoPrime apparatus
Purification of hirudin from its minor isoforms was
performed on a prototype of the IsoPrime [9,10].
Four isoelectric membranes were made having pH values
3.00, 4.00, 4.19, and 5.00. Calculation of the amounts of
buffering and titrant acrylamido compounds was performed
with the program of Giaffreda et al. [11] (Dr. pH, included
with the IsoPrime). All membranes were 10%T, 4%C polyacrylamide gels cast on Whatman GF/D filters, 4.7 cm
diameter, approximately 1 mm thick. After washing the
membranes three times for 20 min each time in distilled
water, the IsoPrime separation unit was assembled and 7 ml
of hirudin was loaded in a single chamber at the anodic side.
The anolyte was 10 mM acetic acid (pH 2.88; conductivity,
85.5 µmhos) and the catholyte 50 mM isoelectric His (pH
7.47; conductivity 67.4 µmhos). Only the contents of anolyte
and catholyte chambers were recycled, those from 200 ml
reservoirs. No circulation reservoirs were connected to the
other separation chambers because of the small amount of
protein processed. Focusing of the minor isoforms of hirudin
was run overnight at 600 V. Joule heat was dissipated in the
cold room (7 °C). Under these conditions, the temperature
rise of the liquid in the separation chamber was only 1 °C at
steady-state.
Absorbance at 220 nm
64
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0
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25
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Time (min)
Figure 4. Limited trypsin proteolysis of HM2 and component b.
Selective trypsin cleavage of HM2 (profile B) gave fragments 1-47
and 48-64 while selective cleavage of component b gave the same
fragment 1-47 and the new fragment 48-59 (profile A). The identification of peptide fragments was confirmed by N-terminal
sequence analysis.
References
1. Dodt, J., (1995) Angew. Chem. Int. Ed. Engl. 34, 867-880.
2. Scacheri, E., Nitti, G., Valsasina, B., Orsini, G., Visco, C., Ferrera, M.,
Sawyer, R.T., Sarmientos, P. (1993) Eur. J. Biochem. 214, 295-304.
3. Anicetti, V.R., Keyt, B.A., Hancock, W.S. (1989) Trends Biotechnol.
7, 342-348.
Ordering Information
4. Markwardt, F. (1957), Hoppe-Seyler Z. Physiol. Chem. 308, 147-156.
5. Eckerskorn, C., Strupat, K., Karas, M., Hillenkamp, F., Lottspeich, F.
(1992) Electrophoresis 13, 664-665.
6. Klarskov, K., Roepstorff, P. (1993) Biol. Mass. Spectrom. 22, 433-440.
7. Dunphy J. C., Busch, K. L., Hettich, R. L., Buchanan, M. V. (1993)
Anal. Chem. 65, 1329-1335.
8. Righetti, P.G. (1990) Immobilized pH Gradients: Theory and
Methodology, Elsevier, Amsterdam,.
9. Righetti, P.G., Wenisch, E., Faupel, M. (1989) J. Chromatogr. 475,
293-309.
10. Righetti, P.G., Wenisch, E., Jungbauer, A., Katinger, H., Faupel, M.
(1990) J. Chromatogr. 500, 681-696.
11. Giaffreda, E., Tonani, C., Righetti, P.G. (1993) J. Chromatogr. 630,
313-327.
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Code No.
Item Description
80-6081-90
80-6082-47
18-1018-06
19-3500-01
80-1255-70
80-1255-71
80-1255-72
80-1255-73
80-1255-74
80-1255-75
IsoPrime Unit, 115 V
IsoPrime Unit, 230 V
Multiphor II Electrophoresis Unit
EPS 3500 XL Power Supply
Immobiline, 10 ml, pk 3.6
Immobiline, 10 ml, pk 4.6
Immobiline, 10 ml, pk 6.2
Immobiline, 10 ml, pk 7.0
Immobiline, 10 ml, pk 8.5
Immobiline, 10 ml, pk 9.3