Corruption of phage display libraries by target-unrelated clones: Diagnosis and countermeasures

Phage display is used to discover peptides or proteins with a desired target property—most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phages or peptides (TUPs), that lack the target behavior. Many TUPs are propagation related; they have mutations conferring a growth advantage and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus-strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus-strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus-strand origin. The founder’s infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption.     

Display of disulfide-rich proteins by complementary DNA display and disulfide shuffling assisted by protein disulfide isomerase

We report an efficient system to produce and display properly folded disulfide-rich proteins facilitated by coupled complementary DNA (cDNA) display and protein disulfide isomerase-assisted folding. The results show that a neurotoxin protein containing four disulfide linkages can be displayed in the folded state. Furthermore, it can be refolded on a solid support that binds efficiently to its natural acetylcholine receptor. Probing the efficiency of the display proteins prepared by these methods provided up to 8-fold higher enrichment by the selective enrichment method compared with cDNA display alone, more than 10-fold higher binding to its receptor by the binding assays, and more than 10-fold higher affinities by affinity measurements. Cotranslational folding was found to have better efficiency than posttranslational refolding between the two investigated methods. We discuss the utilities of efficient display of such proteins in the preparation of superior quality proteins and protein libraries for directed evolution leading to ligand discovery.     

ε-聚赖氨酸生物合成及其产生菌遗传转化研究进展

ε-聚赖氨酸(ε-poly-L-lysine,ε-PL)是由25-35个L-赖氨酸(L-lysine)通过α-ε酰胺键连接的具有很强抗菌活性的聚合物,是自然界中迄今为止仅发现的2种均聚氨基酸(ε-聚赖氨酸和γ-聚谷氨酸)之一。目前,研究发现ε-聚赖氨酸的合成酶是一种非核糖体肽合成酶,它催化前体物质L-lysine经多轮缩合反应合成链长不均一的ε-聚赖氨酸,与I型聚酮合成酶的合成过程相似。ε-聚赖氨酸的合成不受降解酶控制。同时,针对产生菌遗传转化的穿梭质粒载体pLAE001和pLAE003已构建成功,为进一步探索ε-聚赖氨酸生物合成提供了条件。本文主要就ε-聚赖氨酸生物合成及产生菌遗传转化体系进行综述。另外,扼要介绍了作者所在课题组的相关研究工作、取得的进展并提出了相应的见解,论文最后部分对组合生物合成在ε-PL产生菌菌种改造中的应用前景进行了探讨。     

Versatile Solid Supports for Oligonucleotide Synthesis that Incorporate Urea Bridge

The universal solid support, USIII, representing a new and improved version of commercial USII, as well as 2 ′-deoxynucleoside and 2 ′-deoxy-2 ′-fluoronucleoside bound supports, incorporating a labile phenoxyacetyl fragment, was synthesized by an aminomethyl polystyrene carbamoylation with corresponding azides in the presence of aqueous triethylammonium bicarbonate. All three solid phases incorporate a stable urea tether, thus bridging the polymer and functional linker. These new matrices proved to be potent solid phases for the synthesis of DNA, RNA, or modified oligonucleotides as well as randomized mixed 2 ′-ribo/2 ′-deoxy-2 ′-fluoro-RNA libraries and/or DNA libraries, randomized with trinucleotides (codons).     

Isolation and affinity maturation of hapten-specific antibodies

More and more recombinant antibodies specific for haptens such as drugs of abuse, dyes and pesticides are being isolated from antibody libraries. Thereby isolated antibodies tend to possess lower affinity than their parental, full-size counterparts, and therefore the isolation techniques must be optimized or the antibody genes must be affinity-matured in order to reach high affinities and specificities required for practical applications. Several strategies have been explored to obtain high-affinity recombinant antibodies from antibody libraries: At the selection level, biopanning optimization can be performed through elution with free hapten, analogue pre-incubation and subtractive panning. At the mutagenesis level, techniques such as random mutagenesis, bacterial mutator strains passaging, site-directed mutagenesis, mutational hotspots targeting, parsimonious mutagenesis, antibody shuffling (chain, DNA and staggered extension process) have been used with various degrees of success to affinity mature or modify hapten-specific antibodies. These techniques are reviewed, illustrated and compared.     

Engineering a circularly permuted GFP scaffold for peptide presentation

The use of peptides as in vivo and in vitro ligand binding agents is hampered by the high flexibility, low stability and lack of intrinsic detection signal of peptide aptamers. Recent attempts to overcome these limitations included the integration of the binding peptide into a stable protein scaffold. In this paper, we present the optimization and testing of a circularly permuted variant of the green fluorescent protein (GFP). We examined the ability of the optimized scaffold to accept peptide insertions at three different regions. The three regions chosen are localized in close spatial proximity to each other and support different conformations of the inserted peptides. In all the three regions peptides with a biased, but still comprehensive, amino acid repertoire could be presented without disturbing the function of the optimized GFP-scaffold.     

Design and synthesis of new substrates of HtrA2 protease

HtrA2 belongs to the HtrA (high temperature requirement A) family of ATP-independent serine proteases. The primary function of HtrA2 includes maintaining the mitochondria homeostasis, cell death (by apoptosis, necrosis, or anoikis), and contribution to the cell signaling. Several recent reports have shown involvement of HtrA2 in development of cancer and neurodegenerative disorders. Here, we describe the profiling of HtrA2 protease substrate specificity via the combinatorial chemistry approach that led to the selection of novel intramolecularly quenched substrates. For all synthesized compounds, the highest HtrA2-mediated hydrolysis efficiency and selectivity among tested HtrA family members was observed for ABZ-Ile-Met-Thr-Abu-Tyr-Met-Phe-Tyr(3-NO₂)-NH₂, which displayed a specificity constant k_cat/K_M value of 14,535 M⁻¹ s⁻¹.     

Recombinant polyclonal antibodies for cancer therapy

Although monoclonal antibodies are increasingly used for cancer therapy, remissions are only temporary due to emergence of tumor cell escape variants that are no longer affected by the antibody. The emergence of escape variants could be minimized by multi-targeting of tumor cells with polyclonal antibodies, which would also be more efficient than monoclonal antibodies at mediating effector functions for target destruction. A technology for generating recombinant polyclonal antibodies for cancer therapy has been developed based on the construction and selection of tumor-reactive Fab phage display libraries. The selected Fabs are mass-converted to full-length polyclonal antibody libraries (PCALs) of any isotype and any species. Prototypic PCALs generated against human colorectal cancer cell lines showed that libraries of diverse recombinant antibodies, enriched for reactivity to the cancer cells compared to normal human cells, can be obtained. The success of recombinant polyclonal antibodies as cancer therapeutics will depend on the ability to generate, characterize, and mass-produce PCALs with high ratios of cancer-to-normal reactivities that cross-react with many cancers of the same type.