Mingyue He

Protein microarrays: high-throughput tools for proteomics.

Protein microarrays are versatile tools for parallel, miniaturized screening of binding events involving large numbers of immobilized proteins in a time- and cost-effective manner. They are increasingly applied for high-throughput protein analyses in many research areas, such as protein interactions, expression profiling and target discovery. While conventionally made by the spotting of purified proteins, recent advances in technology have made it possible to produce protein microarrays through in situ cell-free synthesis directly from corresponding DNA arrays. This article reviews recent developments in the generation of protein microarrays and their applications in proteomics and diagnostics.

In situ synthesis of protein arrays

In situ or on-chip protein array methods use cell free expression systems to produce proteins directly onto an immobilising surface from co-distributed or pre-arrayed DNA or RNA, enabling protein arrays to be created on demand. These methods address three issues in protein array technology: (i) efficient protein expression and availability, (ii) functional protein immobilisation and purification in a single step and (iii) protein on-chip stability over time. By simultaneously expressing and immobilising many proteins in parallel on the chip surface, the laborious and often costly processes of DNA cloning, expression and separate protein purification are avoided. Recently employed methods reviewed are PISA (protein in situ array) and NAPPA (nucleic acid programmable protein array) from DNA and puromycin-mediated immobilisation from mRNA.

Eukaryotic ribosome display with in situ DNA recovery

Ribosome display is a cell-free technology for the in vitro selection and evolution of proteins encoded by DNA libraries, in which individual nascent proteins (phenotypes) are linked physically to their corresponding mRNA (genotypes) in stable protein-ribosome-mRNA (PRM) complexes (Fig. 1). Formation of the complexes can be achieved through deletion of the stop codon of the mRNA, stalling the ribosome at the end of translation; the nascent protein is extended by a spacer such as the immunoglobulin Cκ domain or others to allow exit through the ribosome tunnel. Through affinity for a ligand, the protein-mRNA coupling permits simultaneous isolation of a functional nascent protein and its translated mRNA; the latter is then converted into cDNA by reverse transcription and amplified for further manipulation, repeated cycles or soluble protein expression. Through the use of PCR-generated libraries, avoiding the need for cloning, ribosome display can be used to both screen very large populations and continuously search for new diversity during subsequent rounds of selection. Additionally, the use of cell-free systems allows the selection of proteins that are toxic or unstable in cells, and proteins with chemical modifications. Ribosome display systems using both prokaryotic and eukaryotic cell extracts have been developed. Examples of the application of eukaryotic systems include the selection and evolution of antibody fragments, DNA binding domains, enzymes, interacting proteins and peptides among others. Here we describe the step-by-step procedure to perform our previously described eukaryotic ribosome display method, which has the distinctive feature of an in situ reverse transcription–PCR (RT-PCR) procedure for DNA recovery from ribosome-bound mRNA. We also introduce a recent, previously unpublished improvement to the procedure in which in situ reverse transcription is combined with sensitive single-primer PCR technology.

DiscernArray™ technology: a cell-free method for the generation of protein arrays from PCR DNA

Protein array technology offers a powerful tool to bridge genomics and proteomics. Currently, the bottleneck in the generation of protein arrays is the comprehensive production of functional proteins. We have developed a rapid cell-free method, DiscernArray, which creates functional protein arrays directly from PCR DNA by in vitro synthesis of individual tagged proteins on tag-binding surfaces, such that the tagged proteins are immobilized on a surface as they are synthesised. DiscernArray is particularly useful for arraying proteins and domains which cannot be functionally produced in heterologous expression systems or for which the cloned DNA is not available.

Ribosome display: next-generation display technologies for production of antibodies in vitro.

Antibodies represent an important and growing class of biologic research reagents and biopharmaceutical products. They can be used as therapeutics in a variety of diseases. With the rapid expansion of proteomic studies and biomarker discovery, there is a need for the generation of highly specific binding reagents to study the vast number of proteins encoded by the genome. Display technologies provide powerful tools for obtaining antibodies. Aside from the preservation of natural antibody repertoires, they are capable of exploiting diversity by DNA recombination to create very large libraries for selection of novel molecules. In contrast to in vivo immunization processes, display technologies allow selection of antibodies under in vitro-defined selection condition(s), resulting in enrichment of antibodies with desired properties from large populations. In addition, in vitro selection enables the isolation of antibodies against difficult antigens including self-antigens, and this can be applied to the generation of human antibodies against human targets. Display technologies can also be combined with DNA mutagenesis for antibody evolution in vitro. Some methods are amenable to automation, permitting high-throughput generation of antibodies. Ribosome display is considered as representative of the next generation of display technologies since it overcomes the limitations of cell-based display methods by using a cell-free system, offering advantages of screening larger libraries and continuously expanding new diversity during selection. Production of display-derived antibodies can be achieved by choosing one of a variety of prokaryotic and eukaryotic cell-based expression systems. In the near future, cell-free protein synthesis may be developed as an alternative for large-scale generation of antibodies.

Modulation of antibody display on M13 filamentous phage

Here we describe a phage vector for the display of single chain antibodies and polypeptides on the surface of filamentous M13 phage which permits facile manipulation of the valency of display. The gene encoding the polypeptide is fused to a synthetic copy of the major coat protein VIII gene (gpVIII) which permits incorporation into the phage during assembly of the filament. Here we examine the growth parameters of phage propagation on the subsequent selection of an anti-progesterone antibody fragment from a mixture of display phage. Our results suggest that the density of the polypeptides displayed on phage may be modulated by altering growth conditions. This ability to influence polypeptide display density on filamentous phage may provide a versatile approach for accessing complex libraries and the capture of weaker ligand receptor interactions by avidity, whilst the potential to access and discriminate between higher affinity interactions is not negated.

Production of Protein Arrays by Cell-Free Systems

Protein arrays make possible the functional screening of large numbers of immobilized proteins in parallel. To facilitate the supply of proteins and to avoid their deterioration on storage, we describe our protein in situ array (PISA) method for production of protein arrays in a single step directly from PCR DNA, using cell-free transcription and translation. In PISA, the in vitro-generated proteins are immobilized, as they are formed, on the surface of wells, beads, or slides coated with a protein-capturing reagent. In our preferred method, proteins are tagged with a double-hexahistidine sequence that binds strongly to Ni-NTA-coated surfaces. Advantages of PISA include avoiding bacterial expression and protein purification and making functional protein arrays available as required from genetic information.

Enhanced cell-free protein expression by fusion with immunoglobulin Cκ domain

While cell‐free systems are increasingly used for protein expression in structural and functional studies, several proteins are difficult to express or expressed only at low levels in cell‐free lysates. Here, we report that fusion of the human immunoglobulin κ light chain constant domain (Cκ) at the C terminus of four representative proteins dramatically improved their production in the Escherichia coli S30 system, suggesting that enhancement of cell‐free protein expression by Cκ fusion will be widely applicable.

Selection of Recombinant Antibodies by Eukaryotic Ribosome Display

Ribosome display is a powerful method for selection of single-chain antibodies in vitro. It operates through the formation of libraries of antibody-ribosome-mRNA complexes that are selected on immobilized antigen, followed by recovery of the genetic information from the mRNA by RT-PCR. Both prokaryotic and eukaryotic versions are used. We describe our eukaryotic system, in which rabbit reticulocyte extracts are used for cell free transcription/translation and cDNA is recovered by in situ RT-PCR performed on the selected complexes.

Accessing of recombinant human monoclonal antibodies from patient libraries by eukaryotic ribosome display

What are effective antibodies and when do they arise to prevent or delay disease onset during a natural infection or in the course of vaccination? To address these questions at a molecular level requires longitudinal studies, capturing and analyzing the antibody repertoire at regular intervals following exposure or sero-conversion. Such studies require a method that allows the rapid generation and evaluation of monoclonal antibodies from relatively small volumes of blood. Here we describe an approach for rapidly generating human monoclonal antibodies in vitro by directly screening single-chain antibody repertories derived from donor peripheral blood mononuclear cells using ribosome display. Two single-chain antibody libraries were constructed using RNA extracted from peripheral blood mononuclear cells of two HIV-1 long-term non-progressor donors (K530 and M325). Both libraries were subjected to a single round of in vitro ribosome display for enrichment of human monoclonal antibodies against recombinant gp120(K530), derived from virus isolated from donor K530. This study has validated a novel, in vitro method for the rapid generation of human monoclonal antibodies. An antibody library could be constructed from as little as 3 μg of total RNA, the equivalent of 3-5 mL of human blood.