Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries

We report for the first time the affinity maturation of Fab antibody fragments using fluorescent-activated cell sorting (FACS) of yeast-displayed repertoires. A single yeast display vector which enables the inducible expression of an anchored heavy chain and a soluble light chain has been constructed. The assembly and functional display on the yeast cell surface of Fab antibodies specific for different protein targets has been demonstrated by flow cytometry and immunofluorescence microscopy. We have affinity matured a Fab antibody specific for the tetravalent antigen streptavidin using FACS of yeast-displayed repertoires diversified by error-prone polymerase chain reaction. A panel of variants with up to 10.7-fold improvement in affinity was obtained after selection. Two leading clones, R2H10 (3.2 nM) and R3B1 (5.5 nM), had mutations in light chain complementarity determining region 1 LC-CDR1 (H34R) and LC-CDR3 (Y96H or Y96F) and gave a 10.7-fold and 6.3-fold affinity improvement over the starting antibody, respectively. The ability to efficiently affinity mature Fab antibodies is an important component of the antibody development pipeline and we have shown that yeast display is an efficient method for this purpose.     

Molecular basis for prostaglandin potency. III. Tests of the significance of the “hairpin conformation” in biorecognition phenomena

Prostaglandin analogs of the PGF_2α, 15-epi-PGF_2α, and PGE₂ type bearing the following methyl substitution patterns — 15-Me, 16, 16-(Me)₂, 17, 17-(Me)₂, and 18, 18, 20-(Me)₃ — and analogs constrained to “hairpin” alignment [via 1, (ω-1)-olide formation] and to “non-hairpin” arrangements [via 1,9- and 1,15-olide formation] are compared in the following biological assays: contraction of uterine and gastro-intestinal smooth muscle strips, luteolytic antifertility potency in the hamster, binding affinity to two different PGF₂α-receptor preparations from bovine corpora lutea, binding to the PGE-specific receptors from rat kidney and liver, inhibition of ADP-induced aggregation of human platelet-rich-plasma, and the effect on rat blood blood pressure. The methylated prostaglandins were also converted to the corresponding prostacyclins and examined as to action on the platelet and on rat blood pressure. All evidence points to topographically distinct receptors for F₂α-, E- and I₂-type prostaglandins. Cross-reactivity is reduced in most of the analogs examined. Independent of the target organ or tissue, the receptors show common features based on the functional class of PG recognized. “Hairpin” alignment improves binding (and potency) only for the PGF₂α specific assays. PGE-specific binding and potency is disrupted to an increasing extent as the chain branching point is moved $\text{further}$ from the 15-hydroxyl center. In contrast 16, 16-dimethylation is particularly disruptive for the PGI₂/E₁platelet receptor interaction.     

Quaternary structural changes in aspartate carbamoyltransferase of Escherichia coli at pH 8.3 and pH 5.8

Atomic coordinates obtained from the crystal structures of unliganded and liganded aspartate carbamoyltransferase at pH 5.8 yield calculated low angle scattering curves in substantial agreement with experimental curves obtained by Moody, Vachette and Foote at pH 8.3. Thus the major conformational changes produced upon binding of six molecules of ligand, N-phosphonacetyl-L-aspartate (PALA) are very similar at pH 8.3 where the enzyme shows activity and regulation, as at pH 5.8 where the enzyme is inactive.     

Protease inhibitor display M13 phage: selection of high-affinity neutrophil elastase inhibitors.

We report display of the complete protease inhibitor (Kunitz) domain, BPTI, on the surface of bacteriophage M13 as a fusion to the gene III product. Phage that display BPTI bind specifically to anti-BPTI antibodies, trypsin and anhydrotrypsin. A point mutation of BPTI [Lys15-->Leu(K15L)] alters the binding specificity of fusion phage such that a human neutrophil elastase-binding phenotype is conferred while a trypsin-binding phenotype is eliminated. Phage were eluted from an immobilized protease with step gradients of decreasing pH. Phage that display Kunitz domains having higher affinity for the immobilized protease exhibit characteristic pH elution phenotypes, indicating that bound display phage can be selectively recovered from an affinity matrix. Utilization of this technology should enable the selection of remodeled protease inhibitors exhibiting novel binding specificities.     

Thermococcus kodakarensis has two functional PCNA homologs but only one is required for viability

Proliferating cell nuclear antigen (PCNA) monomers assemble to form a ring-shaped clamp complex that encircles duplex DNA. PCNA binding to other proteins tethers them to the DNA providing contacts and interactions for many other enzymes essential for DNA metabolic processes. Most eukarya and euryarchaea have only one PCNA homolog but Thermococcus kodakarensis uniquely has two, designated PCNA1 and PCNA2, encoded by TK0535 and TK0582, respectively. Here, we establish that both PCNA1 and PCNA2 form homotrimers that stimulate DNA synthesis by archaeal DNA polymerases B and D and ATP hydrolysis by the replication factor C complex. In exponentially growing cells, PCNA1 is abundant and present at an ~100-fold higher concentration than PCNA2 monomers. Deletion of TK0582 (PCNA2) had no detectable effects on viability or growth whereas repeated attempts to construct a T. kodakarensis strain with TK0535 (PCNA1) deleted were unsuccessful. The implications of these observations for PCNA1 function and the origin of the two PCNA-encoding genes in T. kodakarensis are discussed.