Guidelines for nucleic acid template design for optimal cell-free protein synthesis using an Escherichia coli reconstituted system or a lysate-based system.

Cell-free protein synthesis is an attractive method for generating enzyme/protein variants for simplified functional analysis as both in vitro protein expression and analysis may often be performed in a single vial or well. Today, researchers may choose from multiple commercial cell lysate products or reconstituted systems which are compatible with either mRNA, linear DNA or plasmid DNA templates. Here we provide guidance for optimal design of the genetic elements within linear and plasmid DNA templates which are required to reliably practice cell-free protein synthesis. Protocols are presented for generating linear DNA templates, and data are presented to show that linear DNA templates may in many cases provide robust protein yields even when employing an Escherichia coli lysate for protein synthesis. Finally, the use of linear DNA templates makes it possible to bypass all cell cultivation steps and proceed from PCR amplification of synthetic DNA to generation of target protein in a matter of hours.

Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria.

Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs­named 'cyclonals'­effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation.

Protein synthesis using a reconstituted cell-free system.

Most cell-free protein-synthesis systems are based on cell extracts, which often contain undesirable activities. Reconstituted systems, by contrast, are composed of a defined number of purified and recombinant components with minimal nuclease and protease activities. This unit describes the use of a particular commercial reconstituted system, PURExpress. This system allows in vitro synthesis of proteins from mRNA and circular and linear DNA templates, as well as co-translational labeling of proteins. Unique to this system, all recombinant protein components of the system are His-tagged, allowing purification of the synthesized untagged protein by removing the rest of the system's components. Newly synthesized proteins can often be visible on an SDS-PAGE gel and directly assayed for their functions without labeling and purification. Certain components of the system, such as ribosomes or release factors, can be omitted for specific applications. Such "delta" versions of the system are well suited for studies of bacterial translation, assays of ribosome function, incorporation of unnatural amino acids, and ribosome display of protein libraries.

Selection for mutants improving expression of an anti-MAP kinase monoclonal antibody by filamentous phage display.

It has recently been reported that single amino acid residues can strongly influence the expression of recombinant antibody fragments in Escherichia coli. Prediction of these critical positions can be difficult even with prior knowledge of the primary sequence and the three-dimensional folded structure of the antibody. To circumvent this, a Fab phage display library containing random point mutations was generated from a hybridoma specific for activated p44/p42 mitogen-activated protein (MAP) kinases. Clones that express Fab were selected by panning against the target antigen. It was found that a cysteine-to-serine substitution at position 91 in the CDR3 of the light chain was responsible for allowing expression of Fab. Site-directed mutagenesis was performed to effect this substitution and others at cysteine 91 on a nonexpressing clone. Mutants containing serine, glycine or alanine at position 91 expressed Fab and bound to target antigen. In contrast, tyrosine mutants had moderate Fab expression but no detectable binding to antigen. These results demonstrate that by using phage display, one can select for the expression of antibody fragments while retaining biological activity.