Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants

For the past two decades, therapeutic and industrially important proteins have been expressed in plants with varying levels of success. The two major challenges hindering the economical production of plant-made recombinant proteins include inadequate accumulation levels and the lack of efficient purification methods. To address these limitations, several fusion protein strategies have been recently developed to significantly enhance the production yield of plant-made recombinant proteins, while simultaneously assisting in their subsequent purification. Elastin-like polypeptides are thermally responsive biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the purification of recombinant proteins. Hydrophobins are small fungal proteins capable of altering the hydrophobicity of their respective fusion partner, thus enabling efficient purification by surfactant-based aqueous two-phase systems. Zera, a domain of the maize seed storage protein γ-zein, can induce the formation of protein storage bodies, thus facilitating the recovery of fused proteins using density-based separation methods. These three novel protein fusion systems have also been shown to enhance the accumulation of a range of different recombinant proteins, while concurrently inducing the formation of protein bodies. The packing of these fusion proteins into protein bodies may exclude the recombinant protein from normal physiological turnover. Furthermore, these systems allow for quick, simple and inexpensive nonchromatographic purification of the recombinant protein, which can be scaled up to industrial levels of protein production. This review will focus on the similarities and differences of these artificial storage organelles, their biogenesis and their implication for the production of recombinant proteins in plants and their subsequent purification.     

Oleosin fusion expression systems for the production of recombinant proteins

For the production of recombinant proteins, product purification is potentially difficult and expensive. Plant oleosins are capable of anchoring onto the surface of natural or artificial oil bodies. The oleosin fusion expression systems allow products to be extracted with oil bodies. In vivo, oleosin fusions are produced and directly localized to natural oil bodies in transgenic plant seeds. Via the oleosin fusion technology the thrombin inhibitor hirudin has been successfully produced and commercially used in Canada. In vitro, artificial oil bodies have been used as “carriers” for the recombinant proteins expressed in transformed microbes. In this article, plant oleosins, strategies and limitations of the oleosin fusion expression systems are summarized, alongside with progress and applications. The oleosin fusion expression systems reveal an available way to produce recombinant biopharmaceuticals at large scale.     

Effects of vector fusion peptides on the conformation and immune reactivity of epitope-shuffled, recombinant multi-epitope antigens

The use of multi-epitopes has been considered as a promising strategy to overcome the obstacle of antigenic variation in malarial vaccine development. Previously, we constructed a multi-epitope artificial antigen, Malaria Random Constructed Antigen-1(M.RCAg-1), to optimize expression of the antigen, and we subcloned the gene into three prokaryotic expression vectors that contain different fusion tags at the N-terminus. Three recombinant proteins expressed by these vectors, named M.RCAg-1/Exp.V-1, V-2, and V-3, were purified after the cleavage of the fusion tag. All three recombinant proteins were able to induce similar levels of antigenicity in BALB/c murine models. However, the antibody responses against the individual epitope peptides of the recombinant products were dramatically different. Additionally, the different epitopes elicited various CD4(+) T-cell responses, as shown by the resulting lymphocyte proliferation and varied IFN-γ and IL-4 levels determined by EILSPOT; however, each could be distinctly recognized by sera derived from malaria patients. Additionally, the rabbit antibody induced by these proteins showed diverse efficacy in malaria parasite growth inhibition assays in vitro. Furthermore, analysis via circular dichroism spectroscopy confirmed that the secondary structure was different among these recombinant proteins. These results suggest that the expressed multi-epitope artificial antigens originating from the different vector fusion peptides indeed affect the protein folding and, subsequently, the epitope exposure. Thus, these proteins are able to induce both distinct humoral and cellular immune responses in animal models, and they affect the efficacy of immune inhibition against the parasite. This work should lead to a further understanding of the impact of vector fusion peptides on the conformation and immune reactivity of recombinant proteins and could provide a useful reference for the development of artificial multi-epitope vaccines.     

Recombinant human chromosomal proteins HMG-14 and HMG-17.

Vectors for expressing human chromosomal proteins HMG-14 and HMG-17 in bacterial cultures under the control of the temperature-inducible lambda PL promoter have been constructed. The open reading frames of the cDNAs have been amplified by the polymerase chain reaction (PCR), utilizing amplimers containing desired restriction sites, thereby facilitating precise location of the initiation codon downstream from a ribosomal binding site. Expression of the recombinant proteins does not significantly affect bacterial growth. The rate of synthesis of the recombinant proteins is maximal during the initial stages of induction and slows down appreciably with time. After an initial burst of protein synthesis, the level of the recombinant protein in the bacterial extracts remains constant at different times following induction. Methods for rapid extraction and purification of the recombinant proteins are described. The recombinant proteins are compared to the proteins isolated from eucaryotic cells by electrophoretic mobility, Western analysis and nucleosome core mobility-shift assays. The ability of the proteins to shift the mobility of the nucleosome cores, but not that of DNA, can be used as a functional assay for these HMG proteins. A source for large quantities of human chromosomal proteins HMG-14 and HMG-17 will facilitate studies on their structure, cellular function and mechanism of interaction with nucleosomes.     

A nonradioactive screening method for cloning genes encoding sequence-specific DNA binding proteins.

We have developed a novel nonradioactive screening method for cloning genes encoding sequence-specific DNA binding proteins. This method is derived from previously described protocols developed for the same purpose by using radioactively labeled DNA probes containing protein recognition sequences. This nonradioactive strategy relies upon the use of a small hapten, digoxigenin. Fusion proteins expressed from the recombinant bacteriophage lambda gt11/lambda ZAP are immobilized on nitrocellulose filters and probed with digoxigenin-labeled double-stranded DNA as a ligand. The specifically bound DNA probes can be detected through sequential incubations with antibody-enzyme conjugate and enzyme substrates. This technique has been successfully utilized to isolate several cDNA clones encoding DNA binding proteins.     

Engineering lysine‐rich caleosins as carrier proteins to render biotin as a hapten on artificial oil bodies for antibody production

It has been demonstrated that caleosin alone is sufficient to stabilize artificial oil bodies. A series of recombinant caleosins, mutated with 3, 5, 8, 11, 13, 15, and 17 extra Lys residues and over‐expressed in Escherichia coli, were used as carrier proteins to render biotin as a hapten on the surface of artificial oil bodies for antibody production. Biotinylation levels of the recombinant caleosins were step‐wisely elevated as the number of extra Lys residues increased, and the biotinylated Lys residues were identified by mass spectrometric analysis. Polyclonal antibodies against biotin were successfully generated in rats injected with artificial oil bodies constituted with each of the biotinylated caleosins. Moreover, those generated via the biotinylated caleosins with eight or more extra Lys residues no longer recognized caleosin. It appears that engineered Lys‐rich caleosins are suitable carrier proteins for the production of antibodies against small molecules. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Baculovirus-expressed myogenic determination factors require E12 complex formation for binding to the myosin-light-chain enhancer.

Two recombinant baculoviruses BcV-myf4 and BcV-myf5 have been constructed to synthesize the human myogenic determination factors myogenin (myf4) and myf5 in eucaryotic cells. Both recombinant proteins are localized to the nucleus of virus-infected Spodoroptera frugiperda (sf) insect cells and can be recovered as soluble factors. The virus-produced proteins exhibit high-affinity binding to a muscle-specific DNA sequence in the presence of the ubiquitous helix-loop-helix (HLH) protein E12, but only marginal binding in unsupplemented sf nuclear extracts. Both baculovirus-encoded myogenic factors are able to heterooligomerize with E12 in the absence of DNA-binding sites. We conclude from our results that these muscle-specific HLH proteins produced in eucaryotic cells largely depend on dimerization with E12 or similar HLH proteins to recognize the myosin-light-chain-enhancer-MEF-1-binding site. We have no evidence for intracellular protein modifications exerting major effects on the interaction between these factors and DNA.     

Soluble Expression and Complex Formation of Proteins Required for HCMV DNA Replication Using the SFV Expression System

Several of the viral proteins required for human cytomegalovirus (HCMV) DNA replication have been difficult to study due to their low abundance in infected cells and low solubility in bacterial or insect-cell expression systems. Therefore we used the Semliki Forest virus expression system to express these proteins in mammalian cells. All of the recombinant proteins were soluble, on the basis of ultracentrifugation properties and their ability to be immunoprecipitated from solution with specific antibodies. Pulse-chase analysis of the 86-kDa major immediate-early protein (IE86) revealed two expressed forms—a precursor and a product—indicating that this recombinant protein, like the native HCMV protein, is posttranslationally processed. The recombinant proteins (polymerase core and accessory as well as the IE86 and pUL84) formed stable complexes similar to those known to form in HCMV-infected cells. The recombinant DNA polymerase holoenzyme also exhibited enzyme activity that was phosphonoformic acid sensitive, as is the infected-cell DNA polymerase activity. This expression system offers many advantages for the expression and study of the HCMV replication proteins, including the expression of soluble, active proteins that are able to interact to form complexes. Additionally, the relative ease with which SFV recombinants can be made lends itself to the construction and evaluation of mutants.     

Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis.

We have developed efficient methods for creating artificial transposons and inserting these transposons into plasmid targets in vitro, primarily for the purpose of DNA mapping and sequencing. A novel plasmid has been engineered to convert virtually any DNA sequence, or combination of sequences, into an artificial transposon; hence, custom transposons containing any desired feature can be easily designed and constructed. Such transposons are then efficiently inserted into plasmid targets, in vitro, using the integrase activity present in yeast Ty1 virus-like particles. A single in vitro integration reaction, which resembles a simple restriction digestion in the complexity of the reaction, gives rise to thousands of recoverable insertion events within DNA target molecules; this frequency approaches one insertion per phosphodiester bond in typical plasmids. Importantly, transposon insertions are recovered from all regions of DNA inserts carried on plasmid targets, indicating that integration is a random or nearly-random process. Because of its versatility, this technology offers a generalized method of generating recombinant DNA molecules of a desired structure. We have adapted this system for DNA sequencing by developing a customized artificial transposon to insert new primer binding sites into internal regions of DNA inserts carried on cloning vectors. Transposon insertions have been generated throughout several different yeast and human DNA inserts carried on plasmids, allowing the efficient recovery of sequence information from these inserts. Our results demonstrate the overall utility of this method for both small and large-scale DNA sequencing, as well as general DNA restructuring, and indicate that it could be adapted for use with a number of additional applications including functional genetic analysis.     

融合蛋白基因工程应用及研究

通过重组DNA技术将不同的基因或基因片段融合在一起,经表达后可以得到由不同的功能蛋白拼合在一起而形成的新型多结构域的人工蛋白,这是一种极具潜在应用价值与理论意义的方法。目前这种方法已被广泛应用于许多领域的研究中,并已显示出较高的价值,本文尝试对融合蛋白研究的几个领域的发展情况进行综述。     

Characterization of the DNA-binding properties of the polyomavirus capsid protein VP1.

The major capsid protein of polyomavirus, VP1, has been expression cloned in Escherichia coli, and the recombinant VP1 protein has been purified to near homogeneity (A. D. Leavitt, T. M. Roberts, and R. L. Garcea, J. Biol. Chem. 260:12803-12809, 1985). With this recombinant protein, a nitrocellulose filter transfer assay was developed for detecting DNA binding to VP1 (Southwestern assay). In optimizing conditions for this assay, dithiothreitol was found to inhibit DNA binding significantly. With recombinant VP1 proteins deleted at the carboxy and amino termini, a region of the protein affecting DNA binding was identified within the first 7 amino acids (MAPKRKS) of the VP1 amino terminus. Southwestern analysis of virion proteins separated by two-dimensional gel electrophoresis demonstrated equivalent DNA binding among the different VP1 isoelectric focusing subspecies, suggesting that VP1 phosphorylation does not modulate this function. By means of partial proteolysis of purified recombinant VP1 capsomeres for assessing structural features of the protein domain affecting DNA binding, a trypsin-sensitive site at lysine 28 was found to eliminate VP1 binding to DNA. The binding constant of recombinant VP1 to polyomavirus DNA was determined by an immunoprecipitation assay (R. D. G. McKay, J. Mol. Biol. 145:471-488, 1981) to be 1 x 10(-11) to 2 x 10(-11) M, which was not significantly different from its affinity for plasmid DNA. McKay analysis of deleted VP1 proteins and VP1-beta-galactosidase fusion proteins indicated that the amino terminus was both necessary and sufficient for DNA binding. As shown by electron microscopy, DNA inhibited in vitro capsomere self-assembly into capsidlike structures (D. M. Salunke, D. L. D. Caspar, and R. L. Garcea, Cell 46:895-904, 1986). Thus, VP1 is a high-affinity, non-sequence-specific DNA-binding protein with the binding function localized near its trypsin-accessible amino terminus. The inhibitory effects of disulfide reagents on DNA binding and of DNA on capsid assembly suggest possible intermediate steps in virion assembly.     

Nucleotide sequences of cloned cDNA fragments specific for six Xenopus laevis ribosomal proteins

We have previously constructed and selected six recombinant plasmids containing cDNA sequences specific for different ribosomal proteins of Xenopus laevis (Bozzoni et al., 1981). DNA cloned in these plasmids have been isolated and sequenced. Amino acid sequences of the corresponding portions of the proteins have been derived from DNA sequences; they are arginine- and lysine-rich as expected for ribosomal proteins. One of the cDNA sequences has an open reading frame also on the strand complementary to the one coding for the ribosomal protein; this fragment has inverted repeats twenty nucleotides long at the two ends. The codon usage for the six sequences appears to be non-random with some differences among the ribosomal proteins analysed.     

Enhancement of phage-mediated gene transfer by nuclear localization signal

The cell membrane and the nuclear membrane are two major barriers hindering the free movement of various macromolecules through animal cells. Nevertheless, some proteins can actively bypass these barriers by dint of intrinsic peptidic signals, so incorporation of these signals might improve the efficacy of artificial gene delivery vehicles. We examined the role of the nuclear localization signal (NLS) in gene transfer, using recombinant lambda phage as a model of the polymer/DNA complexes. We prepared a lambda phage displaying a 32-mer NLS of SV40 T antigen on its surface (NLS phage), and found that this NLS phage, delivered into the cytoplasm by appropriate devices, has higher affinity for the nucleus and induces the expression of encapsulated marker genes more efficiently than does the wild-type phage. This suggests that the 32-mer NLS peptide will become a practical tool for artificial gene delivery vehicles with enhanced nuclear targeting activity.     

Recombinant DNA in Medicine

Studies in bacteria and bacterial viruses have led to methods to manipulate and recombine DNA in unique and reproducible ways and to amplify these recombined molecules millions of times. Once properly identified, the recombinant DNA molecules can be used in various ways useful in medicine and human biology. There are many applications for recombinant DNA technology. Cloned complementary DNA has been used to produce various human proteins in microorganisms. Insulin and growth hormone have been extensively and successfully tested in humans and insulin has been licensed for sale. Mass production of bacterial and viral antigens with recombinant DNA technology is likely to provide safe and effective vaccines for some disorders for which there is no prevention. The cloned probes for the human α- and β-globin loci, for specific disease genes, such as the Z allele of α-antitrypsin, and for random genomic sequences are proving useful for prenatally diagnosing human genetic disorders and preventing their clinical consequences.     

ASSEMBLY AND OPTIMIZATION OF EXPRESSION OF SYNTHETIC GENES DERIVED FROM THE HUMAN ELASTIN REPEATED MOTIF

Naturally occurring proteins often possess interesting properties that make them attractive for the realization of innovative biomaterials. Repetitive artificial polypeptides have been modeled on the repeated domains of mammalian elastin, retaining and even enhancing their thermally responsive behavior. These protein polymers have been produced by recombinant biotechnology and due to their smart properties show a huge potential for a wide range of biomedical and biotechnological applications. For this reason, production of large quantities of highly purified material is a crucial step. We focused our attention on elastin-derived polypeptides based on the hexapeptidic motif typical of human elastin. Synthetic genes were assembled starting from a monomeric unit, and the different conditions were assayed to optimize the yield of the artificial polypeptides. Optimization of Escherichia coli strain and of the extraction procedure led to significant improvement in expression and recovery of the recombinant products. Electron micrographs of expressing bacteria under optimized conditions showed the accumulation of the recombinant product in the induced cells.     

蛋白微阵列研究进展

蛋白微阵列是随着基因微阵列技术发展起来的,用于基因微阵列的制备方法、信号的检测及分析系统,也可用于蛋白微阵列。各种蛋白微阵列基质的发展,提高了蛋白的固定效率。放射性同位数、化学发光、激光共聚焦荧光扫描等技术都已用于微阵列的检测。重组蛋白技术的发展,提高了蛋白微阵列检测的通量和灵敏度。蛋白微阵列具有通量高、使用样品少、重复性好、可定量的特点,使其在生物医药科学研究中得到了广泛应用。本综述了蛋白微阵列的制备及其在免疫检测、医学诊断及蛋白组研究中的应用。     

Human artificial chromosomes with alpha satellite-based de novo centromeres show increased frequency of nondisjunction and anaphase lag

Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P < 0.001). Naturally occurring, but abnormal small ring chromosomes derived from chromosome 17 and the X chromosome also missegregate more than normal chromosomes, implicating overall chromosome size and/or structure in the fidelity of chromosome segregation. As different artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes.     

Expression, purification, and physical properties of recombinant flavocytochrome fusion proteins containing rat cytochrome b(5) linked to NADPH-cytochrome P450 reductase by different membrane-binding segments

Reconstitution of the enzymatic activities using purified microsomal cytochrome P450s (P450) requires the presence of a membrane-binding segment in the mammalian flavoprotein, NADPH--cytochrome P450 reductase (CPR), and the hemeprotein, cytochrome b(5) (b(5)). The mechanism(s) by which the membrane-binding segments of these proteins exert such a critical role in influencing the reconstitution of the NADPH-supported activity of a P450 remains undefined. In the present work we describe the construction, expression, and purification of four different types of recombinant flavocytochromes containing rat b(5) and rat CPR linked by various membrane-binding segments. The physical properties of these artificial fusion proteins have been studied to determine their ability to serve as electron transfer agents. These studies are a prelude to the subsequent study (accompanying paper) evaluating the functional roles of the hydrophobic (membrane-binding) sequences of b(5) and CPR in the reconstitution of P450 activities. The present study shows that the purified recombinant fusion proteins can serve as active electron transport carriers from NADPH to cytochrome c as well as b(5) by intramolecular as well as intermolecular reactions. It is shown here that the electron transport properties of these purified fusion proteins are influenced by high concentrations of KCl, suggesting a role for charged amino acids in protein-protein interactions. The present study illustrates the application of artificial recombinant flavocytochromes as useful proteins for the study of intramolecular electron transport reactions for comparison with intermolecular interactions.     

Blueprint for a high-performance biomaterial: full-length spider dragline silk genes

Spider dragline (major ampullate) silk outperforms virtually all other natural and manmade materials in terms of tensile strength and toughness. For this reason, the mass-production of artificial spider silks through transgenic technologies has been a major goal of biomimetics research. Although all known arthropod silk proteins are extremely large (>200 kiloDaltons), recombinant spider silks have been designed from short and incomplete cDNAs, the only available sequences. Here we describe the first full-length spider silk gene sequences and their flanking regions. These genes encode the MaSp1 and MaSp2 proteins that compose the black widow's high-performance dragline silk. Each gene includes a single enormous exon (>9000 base pairs) that translates into a highly repetitive polypeptide. Patterns of variation among sequence repeats at the amino acid and nucleotide levels indicate that the interaction of selection, intergenic recombination, and intragenic recombination governs the evolution of these highly unusual, modular proteins. Phylogenetic footprinting revealed putative regulatory elements in non-coding flanking sequences. Conservation of both upstream and downstream flanking sequences was especially striking between the two paralogous black widow major ampullate silk genes. Because these genes are co-expressed within the same silk gland, there may have been selection for similarity in regulatory regions. Our new data provide complete templates for synthesis of recombinant silk proteins that significantly improve the degree to which artificial silks mimic natural spider dragline fibers.