A general method for the selection of high-level scFv and IgG antibody expression by stably transfected mammalian cells

The isolation of mammalian cell lines capable of high-yield expression of recombinant antibodies is typically performed by screening multiple individual clones by limiting dilution techniques. A number of experimental strategies have recently been devised to identify high-expressing clones, but protocols are often difficult to implement, time consuming, costly and limited in terms of number of clones which can be screened. In this article, we describe new vectors for the expression of recombinant antibodies in IgG format and in other formats, based on the single-chain Fv module, as well as a high-throughput screening procedure, based on the direct staining of antibodies transiting the membrane of a stably transfected cell, followed by preparative sorting using a high-speed cell sorter. This procedure allows, in one step, to deposit single cells into individual wells of a 96-well microtiter plate (thus facilitating cloning) and to preferentially recover those rare cell populations which express dramatically higher levels of recombinant antibody. Using cell cultures followed by affinity purification techniques, we could confirm that the new vectors and the new screening procedure reliably yield high-expression clones and homogenous protein preparations. We expect that these techniques should find broad applicability for both academic and industrial antibody engineering researc

Substrate Specificity of Cysteine Proteases Beyond the S2 Pocket: Mutagenesis and Molecular Dynamics Investigation of Fasciola hepatica Cathepsins L.

Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S2 subsite (pocket) of the enzyme that interacts with the substrate P2 residue (Schetcher and Berger nomenclature). Although secreted FhCL1 accommodates aliphatic residues in the S2 pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S2 subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P3 specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S2 and S3 subsites that contributes to the accommodation of Gly in S3. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S2 and S3 pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity

Substrate Specificity of Cysteine Proteases Beyond the S2 Pocket: Mutagenesis and Molecular Dynamics Investigation of Fasciola hepatica Cathepsins L

Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S2 subsite (pocket) of the enzyme that interacts with the substrate P2 residue (Schetcher and Berger nomenclature). Although secreted FhCL1 accommodates aliphatic residues in the S2 pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S2 subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P3 specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S2 and S3 subsites that contributes to the accommodation of Gly in S3. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S2 and S3 pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity

Cryo-EM structures of the TMEM16A calcium-activated chloride channel

Calcium-activated chloride channels (CaCCs) encoded by TMEM16A(1-3) control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system(4-7). To understand how CaCCs mediate and control anion permeation to fulfil these physiological functions, knowledge of the mammalian TMEM16A structure and identification of its pore-lining residues are essential. TMEM16A forms a dimer with two pores(8,9). Previous CaCC structural analyses have relied on homology modelling of a homologue (nhTMEM16) from the fungus Nectria haematococca that functions primarily as a lipid scramblase(10-12), as well as subnanometre-resolution electron cryo-microscopy(12). Here we present de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol as determined by single-particle electron cryo-microscopy. These structures reveal the ion permeation pore and represent different functional states. The structure in lauryl maltose neopentyl glycol has one Ca2+ ion resolved within each monomer with a constricted pore; this is likely to correspond to a closed state, because a CaCC with a single Ca2+ occupancy requires membrane depolarization in order to open (C.J.P.etal., manuscript submitted). The structure in nanodiscs has two Ca2+ ions per monomer and its pore is in a closed conformation; this probably reflects channel rundown, which is the gradual loss of channel activity that follows prolonged CaCC activation in 1 mM Ca2+. Our mutagenesis and electrophysiological studies, prompted by analyses of the structures, identified ten residues distributed along the pore that interact with permeant anions and affect anion selectivity, as well as seven porelining residues that cluster near pore constrictions and regulate channel gating. Together, these results clarify the basis of CaCC anion conduction

DataSheet1_Substrate Specificity of Cysteine Proteases Beyond the S2 Pocket: Mutagenesis and Molecular Dynamics Investigation of Fasciola hepatica Cathepsins L.XLS

<p>Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S₂ subsite (pocket) of the enzyme that interacts with the substrate P₂ residue (Schetcher and Berger nomenclature). Although secreted FhCL1 accommodates aliphatic residues in the S₂ pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S₂ subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P_2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P₃ specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S₂ and S₃ subsites that contributes to the accommodation of Gly in S₃. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S₂ and S₃ pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity.</p

Table1_Substrate Specificity of Cysteine Proteases Beyond the S2 Pocket: Mutagenesis and Molecular Dynamics Investigation of Fasciola hepatica Cathepsins L.DOCX

<p>Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S₂ subsite (pocket) of the enzyme that interacts with the substrate P₂ residue (Schetcher and Berger nomenclature). Although secreted FhCL1 accommodates aliphatic residues in the S₂ pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S₂ subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P_2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P₃ specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S₂ and S₃ subsites that contributes to the accommodation of Gly in S₃. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S₂ and S₃ pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity.</p

Image1_Substrate Specificity of Cysteine Proteases Beyond the S2 Pocket: Mutagenesis and Molecular Dynamics Investigation of Fasciola hepatica Cathepsins L.PDF

<p>Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S₂ subsite (pocket) of the enzyme that interacts with the substrate P₂ residue (Schetcher and Berger nomenclature). Although secreted FhCL1 accommodates aliphatic residues in the S₂ pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S₂ subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P_2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P₃ specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S₂ and S₃ subsites that contributes to the accommodation of Gly in S₃. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S₂ and S₃ pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity.</p

Table2_Substrate Specificity of Cysteine Proteases Beyond the S2 Pocket: Mutagenesis and Molecular Dynamics Investigation of Fasciola hepatica Cathepsins L.pdf

<p>Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S₂ subsite (pocket) of the enzyme that interacts with the substrate P₂ residue (Schetcher and Berger nomenclature). Although secreted FhCL1 accommodates aliphatic residues in the S₂ pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S₂ subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P_2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P₃ specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S₂ and S₃ subsites that contributes to the accommodation of Gly in S₃. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S₂ and S₃ pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity.</p