RESUMO
Measurement of intracellular calcium in live cells is a key component of a wide range of basic life science research, and crucial for many high-throughput assays used in modern drug discovery. Synthetic calcium indicators have become the industry standard, due their ease of use, high reliability, wide dynamic range, and availability of a large variety of spectral and chemical properties. Genetically-encoded calcium indicators (GECIs) have been optimized to the point where their performance rivals that of synthetic calcium indicators in many applications. Stable expression of a GECI has distinct advantages over synthetic calcium indicators in terms of reagent cost and simplification of the assay process. We generated a clonal cell line constitutively expressing GCaMP6s; high expression of the GECI was driven by coupling to a blasticidin resistance gene with a self-cleaving cis-acting hydrolase element (CHYSEL) 2A peptide. Here, we compared the performance of the GECI GCaMP6s to the synthetic calcium indicator fluo-4 in a variety of assay formats. We demonstrate that the pharmacology of ion channel and GPCR ligands as determined using the two indicators is highly similar, and that GCaMP6s is viable as a direct replacement for a synthetic calcium indicator.
Assuntos
Bioensaio , Sinalização do Cálcio , Cálcio/análise , Compostos de Anilina/química , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Células HEK293 , Humanos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Xantenos/químicaRESUMO
Humanization of a potent neutralizing mouse anti-human IL-13 antibody (m836) using a method called human framework adaptation (HFA) is reported. HFA consists of two steps: human framework selection (HFS) and specificity-determining residue optimization (SDRO). The HFS step involved generation of a library of m836 antigen binding sites combined with diverse human germline framework regions (FRs), which were selected based on structural and sequence similarities between mouse variable domains and a repertoire of human antibody germline genes. SDRO consisted of diversifying specificity-determining residues and selecting variants with improved affinity using phage display. HFS of m836 resulted in a 5-fold loss of affinity, whereas SDRO increased the affinity up to 100-fold compared to the HFS antibody. Crystal structures of Fabs in complex with IL-13 were obtained for m836, the HFS variant chosen for SDRO, and one of the highest-affinity SDRO variants. Analysis of the structures revealed that major conformational changes in FR-H1 and FR-H3 occurred after FR replacement, but none of them had an evident direct impact on residues in contact with IL-13. Instead, subtle changes affected the V(L)/V(H) (variable-light domain/variable-heavy domain) interface and were likely responsible for the 5-fold decreased affinity. After SDRO, increased affinity resulted mainly from rearrangements in hydrogen-bonding pattern at the antibody/antigen interface. Comparison with m836 putative germline genes suggested interesting analogies between natural affinity maturation and the engineering process that led to the potent HFA anti-human IL-13 antibody.