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Technical Briefs |
Spectral Diagnostics, Inc., 135-2 The West Mall, Toronto ON M9C 1C2, Canada
a author for
correspondence: fax 416-626-3651, e-mail sliu{at}spectraldiagnostics.com
Cardiac troponins are considered the preferred cardiac markers for diagnosis of acute myocardial infarction (1). The development and evaluation of cardiac troponin I immunoassays require purified and immunochemically stable antigen for the production of calibrators and controls. Cardiac troponin I (cTnI) purified from human heart has traditionally been used for such purposes. However, cTnI is unstable and prone to degradation during the process of purification from human heart tissue. Recombinant protein is favored over the protein purified from tissue because it is easier to purify, safer to handle, and has unlimited availability and good reproducibility. In physiological conditions, troponin C (TnC) and TnI along with troponin T (TnT) form an integral protein, designated as troponin complex. To study the immunoactivity of TnI in a clinically relevant form, it is imperative to put TnI in the context of the other two subunits, TnC and TnT.
In this study, human cTnI, cTnC, and cTnT cDNAs were amplified
from the Human Heart Quick-Clone cDNA (Clontech), using
gene-specific primers designed from published cDNA sequences
(2)(3)(4)(5) by PCR. Initially, the cDNA sequence for human
cTnI was inserted into a pET vector without modification. Very little
cTnI was expressed with this construct. To increase the expression of
human cTnI in bacterial host cells, three different modifications were
made to the cDNA sequences separately. The first modified clone,
designated as rcTnI-0, was prepared with two
NH2-terminal codon mutations without changing any
amino acid residues, as described by Al-Hillawi et al. (6).
The second and third modified clones, designated as rcTnI-6 and
rcTnI-14, were constructed with added bacteria-favored
NH2-terminal leader sequences (7); the
leader sequences for rcTnI-6 and rcTnI-14 are MASMGS and
MASMTGGQQMGRGS, respectively. The human cTnT cDNA sequence was modified
as described previously (8) to increase its expression in
Escherichia coli. TnC was expressed without modification to
the cDNA sequence. All constructs were confirmed by DNA sequencing.
Expression constructs for each protein were engineered, transformed,
and expressed according to Shi et al. (9). The purification
processes for these expressed proteins were essentially described by
Shi et al. (9). The concentrations of all troponin subunits
after purification were determined by Bradford assay (10),
with bovine serum albumin as the calibrator. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis was performed on all
samples according to Laemmli (11). As shown in Fig. 1
, human cardiac troponin subunits C, T, and I (I-0, I-6, and
I-14) were successfully expressed in E. coli and purified to
homogeneity. The apparent molecular weights of troponin I-0,
I-6, and I-14 were ~29 000, 30 000, and 31 000, respectively.
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After purification, rcTnI-0, rcTnI-6, and rcTnI-14 were complexed with rcTnC at a 1:1 molar ratio to form troponin CI-0, CI-6, and CI-14 complexes according to Tobacman and Lee (12). Troponin CTI-0, CTI-6, and CTI-14 complexes were also formed by combining subunits C and T with the corresponding subunit I at a molar ratio of 1:1:1 (12). These recombinant polypeptides and their complexes were then tested with the Stratus® TnI immunoassay (13), the Access® TnI immunoassay (14), and a full-sandwich ELISA with goat anti-cTnI polyclonal antibody (15) as the capture antibody and a combination of monoclonal 8I-7 and 2I-14 (15) labeled with horseradish peroxidase as the detector.
The Stratus, Access, and ELISA measurements of these various
troponin forms are summarized in Table 1
. The data show that rcTnI-0, rcTnI-6, and rcTnI-14 are
significantly different from each other in terms of immunoactivity as
determined by the Stratus and Access assays. In the Stratus assay, the
differences in immunoactivity among the free states of rcTnI-0,
rcTnI-6, and rcTnI-14 were larger than those of their binary and
ternary complexes. However, in the Access assay, the differences in
immunoactivity among the free states of rcTnI-0, rcTnI-6, and rcTnI-14
were comparable to those of their binary and ternary complexes.
Furthermore, Access does not recognize rcTnI-14 and its binary and
ternary complexes. The immunoactivities of rcTnI-6 and its complexes
were less than those of rcTnI-0 and its complexes as assessed by
Access. When evaluated by ELISA, the differences among rcTnI-0,
rcTnI-6, rcTnI-14, and their complexes were less distinct.
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The observed differences in immunoactivity among rcTnI-0, rcTnI-6, rcTnI-14, and their complexes can be interpreted as changes in the accessibility of given epitopes of the rcTnIs resulting from the addition of leader sequences. However, the fact that the concentrations of rcTnI-0, rcTnI-6, and rcTnI-14 were determined by Bradford assay needs to be considered when drawing conclusions because the molecular weights of the three rcTnIs are slightly different. In other words, the number of molecules is not exactly the same for a given concentration (by weight per volume) of the three rcTnIs, thus affecting immunoactivity. Based on the molecular weights, 1 µg of rcTnI-0, rcTnI-6, or rcTnI-14 is equal to 41.656, 40.698, or 39.390 pmol, respectively. Therefore, <6% of the differences in immunoactivity among rcTnI-0, rcTnI-6, and rcTnI-14 can be attributed to the use of the Bradford assay. The observed immunoactivity differences among rcTnI-0, rcTnI-6, and rcTnI-14 were much greater than 6%. In the Access assay, immunoactivity was markedly decreased if a leader sequence was present in the TnI molecule. It also seemed that the extent to which immunoactivity was decreased was related to the length of the leader sequence. This tendency was not observed in the Stratus assay, indicating that the presence of leader sequence may affect only certain epitopes and, therefore, certain assays.
It is common practice to add a leader or tag sequence to recombinant proteins to increase expression (7) or to facilitate purification of the expressed product (16). It was not clear whether the leader, the tag, or both would affect the folding status and, thus, the immunoactivity of the resulting recombinant protein (7). Our results suggest that the immunoactivity of a fusion protein can be altered, although the detectability of this alteration may depend on the specific antibodies used.
In conclusion, we have demonstrated for the first time that fused leader sequences affect the immunoactivity of a recombinant protein, presumably by changing its folding status. Therefore, in making controls and calibrators for an immunoassay, the unfused original sequence is highly preferred.
Acknowledgments
We thank Henry T. Burke and Michael Mingfu Ling for their critical review and valuable discussions.
References
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