We observed a gain of motor function with both of the hallmarks in the chimeric prestin without loss of transport function. Motility and nonlinear capacitance were measured from chimeric prestin-transfected human embryonic kidney 293 cells using a voltage-clamp technique and photodiode-based displacement measurement system. To determine whether this sequence represents a motif that facilitates motor function in eutherian prestin, we utilized a chimeric approach by swapping corresponding residues from the zebrafish and chicken with those of gerbil. We identified a segment of 11 amino acid residues in eutherian prestin that is extremely conserved among eutherian species but highly variable among non-mammalian orthologs and SLC26A paralogs. Evidence suggests that prestin orthologs from zebrafish and chicken are anion exchangers or transporters with no motor function. Prestin is unique in this family in that it functions as a voltage-dependent motor protein manifested by two hallmarks, nonlinear capacitance and motility. Prestin belongs to an anion transporter family, the solute carrier protein 26A (SLC26A). Prestin-based OHC motility is thought to be responsible for cochlear amplification, which contributes to the exquisite frequency selectivity and sensitivity of mammalian hearing. This so-called electromotility is the result of conformational changes of membrane-bound prestin. In this case, the C-terminal Lysine residues of the heavy chains can be removed, resulting in variability of the C-terminus of the heavy chains.Ĭontact BioPharmaSpec to find out more about our terminal sequencing services.Cochlear outer hair cells (OHCs) alter their length in response to transmembrane voltage changes. Monoclonal antibodies are an example of proteins where C-terminal variation is observed. Intact molecular weight analysis of the product can also be used as an orthogonal procedure to support conclusions regarding the structures of the N- and C-termini. This includes the generation of confirmatory peptide fragment ions to confirm the nature of the C-terminal peptide or peptides. In the latter approach, intactness of a protein C-terminus or the presence of ragged ends can be assessed using data obtained from a peptide map. For an assessment of the C-termini, BioPharmaSpec scientists use carboxypeptidase digestion and/or mass-spectrometric mapping strategies. There is no fully analogous method akin to Edman chemistry for determining the C-terminal sequencing of a biopharmaceutical. Leucine and isoleucine derivatives have unique chromatographic elution positions and can thus be categorically identified. In these cases, peptides containing Leucine or Isoleucine can be purified and subjected to N-terminal sequencing using Edman chemistry as described above. However, since Leucine and Isoleucine are isomers (they both have the same mass), they cannot be categorically identified by mass. Mass spectrometry can provide much useful structural information as part of an investigation of this nature by generating amino acid sequence information from chromatographically separated peptides. Techniques must be used that can unambiguously define the sequence. As mentioned above, it is a regulatory requirement for the protein sequence to be determined (see Q6B section 6.1.1 a)). This last use of N-terminal sequencing is very important. Mass Spectrometry cannot be used in this instance because there is no mass difference between the two amino acids (they are isomers of one another).Unambiguously defining the Isoleucine and Leucine residues within the protein sequence.Demonstrating batch-to-batch consistency.Showing that the N-terminus of your protein is intact and as expected.N-terminal sequence analysis has a number of different applications in drug development such as:
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