E unum pluribus: multiple proteins from a self-processing polyprotein

Many applications of genetic engineering require transformation with multiple (trans)genes, although to achieve these using conventional techniques can be challenging. The 2A oligopeptide is emerging as a highly effective new tool for the facile co-expression of multiple proteins in a single transformation step, whereby a gene encoding multiple proteins, linked by 2A sequences, is transcribed from a single promoter. The polyprotein self-processes co-translationally such that each constituent protein is generated as a discrete translation product. 2A functions in all the eukaryotic systems tested to date and has already been applied, with great success, to a broad range of biotechnological applications: from plant metabolome engineering to the expression of T-cell receptor complexes, monoclonal antibodies or heterodimeric cytokines in animals.     

Transgene stacking in plants in the absence of sexual crossing

A transgene stacking system is a prerequisite for the introduction of multiple genes and for the modification of complex metabolic pathways in plants. We demonstrate here that the MAT-vector system previously used for generating marker-free transgenic plants is also an efficient and reliable transformation system for the repeated introduction of multiple transgenes independent of sexual crossing. We previously reported that the GST-MAT vector system, in which excision of the yeast site-specific recombination R/RS system is regulated by the maize GST-II-27 promoter, could generate marker-free transgenic plants containing a single transgene with high frequency. Here we show that the GST-MAT vector can be used successfully to introduce a second transgene (GFP) into a marker-free transgenic tobacco line containing single copies of the first transgenes (nptII and uidA genes). The transgene-stacked marker-free transgenic tobacco plants were generated from ca. 20% of excision-positive ipt-shooty explants within 5 months of Agrobacterium infection. The presence of uidA, nptII, GFP genes and the absence of the ipt gene were verified by PCR analyses. Furthermore, Southern blot analysis showed that no chromosomal rearrangements were introduced between the first and second transformations.     

Construction of a new recombinant protein expression system in the basidiomycetous yeast Cryptococcus sp. strain S-2 and enhancement of the production of a cutinase-like enzyme

Yeast host–vector systems have been very successful in expressing recombinant proteins. However, because there are some proteins that cannot be expressed with existing systems, there is a need for new yeast expression systems. Here we describe a new host–vector system based on the basidiomycetous yeast Cryptococcus sp. strain S-2 (S-2). Two advantages of S-2 are that it naturally produces some very useful enzymes, so it would be a good system for expressing multiple copies of some of its genes, and that, it is a nonhazardous species. The orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) gene (URA5) was selected as a selectable marker for transformation in the new host–vector system. URA5 was isolated and introduced into a uracil auxotroph of S-2 by electroporation. To demonstrate the S-2 system, we selected one of its unique enzymes, a plastic-degrading cutinase-like enzyme (CLE). We were able to insert multiple copies of the CLE gene (CLE1) into the chromosomes in a high fraction of the targeted cells. Under optimal conditions, one transformant exhibited 3.5 times higher CLE activity than the wild type. Expression vectors, including an inducible promoter (the promoter for the xylanase or α-amylase gene), were constructed for recombinant protein production, and green fluorescent protein was expressed under the control of these promoters. The xylanase promoter was more tightly controlled. Furthermore, putting CLE1 under the control of the xylanase promoter, which is induced by xylose, increased CLE activity of the culture medium to approximately 15 times greater than that of the wild type.     

Systematic identification of novel, essential host genes affecting bromovirus RNA replication

Positive-strand RNA virus replication involves viral proteins and cellular proteins at nearly every replication step. Brome mosaic virus (BMV) is a well-established model for dissecting virus-host interactions and is one of very few viruses whose RNA replication, gene expression and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. Previously, our laboratory identified ～100 non-essential host genes whose loss inhibited or enhanced BMV replication at least 3-fold. However, our isolation of additional BMV-modulating host genes by classical genetics and other results underscore that genes essential for cell growth also contribute to BMV RNA replication at a frequency that may be greater than that of non-essential genes. To systematically identify novel, essential host genes affecting BMV RNA replication, we tested a collection of ～900 yeast strains, each with a single essential gene promoter replaced by a doxycycline-repressible promoter, allowing repression of gene expression by adding doxycycline to the growth medium. Using this strain array of ～81% of essential yeast genes, we identified 24 essential host genes whose depleted expression reproducibly inhibited or enhanced BMV RNA replication. Relevant host genes are involved in ribosome biosynthesis, cell cycle regulation and protein homeostasis, among other cellular processes. BMV 2a(Pol) levels were significantly increased in strains depleted for a heat shock protein (HSF1) or proteasome components (PRE1 and RPT6), suggesting these genes may affect BMV RNA replication by directly or indirectly modulating 2a(Pol) localization, post-translational modification or interacting partners. Investigating the diverse functions of these newly identified essential host genes should advance our understanding of BMV-host interactions and normal cellular pathways, and suggest new modes of virus control.     

Recombinant baculovirus-based multiple protein expression platform for Drosophila S2 cell culture

A platform for selective and controllable expression of multiple foreign protein types was developed in insect cell culture. Based on the fact that baculovirus cannot replicate in nonpermissive Drosophila melanogaster Schneider line 2 (S2) cells, S2 cells that stably express human erythropoietin (hEPO) under the control of the S2-derived inducible metallothionein (MT) promoter were infected with three types of recombinant baculoviruses, each of which expressed a different fluorescent protein gene under the control of MT promoter. Addition of copper sulfate as an inducer to infected, stably transfected S2 cells resulted in simultaneous expression of hEPO and three fluorescent proteins. Expression profiles and levels of the three induced fluorescent proteins were similar in all single infected cells. Importantly, expression profiles and levels of hEPO were similar in both non-infected and infected cells, indicating that baculovirus expressed recombinant proteins do not adversely affect expression of host cell recombinant proteins. Expressions of the three fluorescent proteins were able to be selectively regulated by altering combination ratios of the three types of recombinant baculoviruses. Collectively, these data indicate that the baculovirus/stably transfected S2 cell system can be successfully used to express multiple foreign proteins in a controlled and selective manner without the burden of additional selection markers. Such a system would be expected to be attractive as a multiple protein expression platform for engineering metabolic or glycosylation pathways.     

Simultaneous Expression of Multiple Proteins Under a Single Promoter in Caenorhabditis elegans via a Versatile 2A-Based Toolkit

Caenorhabditis elegans is a powerful in vivo model in which transgenesis is highly developed. However, while the analysis of biological phenomena often require the expression of more than one protein of interest, no reliable tool exists to ensure efficient concomitant and equivalent expression of more than two polypeptides from a single promoter. We report the use of viral 2A peptides, which trigger a “ribosomal-skip” or “STOP&GO” mechanism during translation, to express multiple proteins from a single vector in C. elegans. Although none of the viruses known to infect C. elegans contain 2A-like sequences, our results show that 2A peptides allow the production of separate functional proteins in all cell types and at all developmental stages tested in the worm. In addition, we constructed a toolkit including a 2A-based polycistronic plasmid and reagents to generate 2A-tagged fosmids. 2A peptides constitute an important tool to ensure the delivery of multiple polypeptides in specific cells, enabling several novel applications such as the reconstitution of multi-subunit complexes.     

Coordinate expression and independent subcellular targeting of multiple proteins from a single transgene

A variety of conventional methods allow the expression of multiple foreign proteins in plants by transgene stacking or pyramiding. However, most of these approaches have significant drawbacks. We describe a novel alternative, using a single transgene to coordinate expression of multiple proteins that are encoded as a polyprotein capable of dissociating into component proteins on translation. We demonstrate that this polyprotein system is compatible with the need to target proteins to a variety of subcellular locations, either cotranslationally or posttranslationally. It can also be used to coordinate the expression of selectable marker genes and effect genes or to link genes that are difficult to assay to reporter genes that are easily monitored. The unique features of this polyprotein system are based on the novel activity of the 2A peptide of Foot-and-mouth disease virus (FMDV) that acts cotranslationally to effect a dissociation of the polyprotein while allowing translation to continue. This polyprotein system has many applications both as a research tool and for metabolic engineering and protein factory applications of plant biotechnology.     

Engineering the plastid genome of higher plants

The plastid genome of higher plants is an attractive target for engineering because it provides readily obtainable high protein levels, the feasibility of expressing multiple proteins from polycistronic mRNAs and gene containment through the lack of pollen transmission. A chloroplast-based expression system that is suitable for the commercial production of recombinant proteins in tobacco leaves has been developed recently. This expression system includes vectors, expression cassettes and site-specific recombinases for the selective elimination of marker genes. Progress in expressing proteins that are biomedically relevant, in engineering metabolic pathways, and in manipulating photosynthesis and agronomic traits is discussed, as are the problems of implementing the technology in crops.