Review and patents and literature. The use of insect cell cultures for recombinant protein synthesis: Engineering aspects.

The use of the insect cell/baculovirus expression system for producing recombinant proteins of bacterial, plant, insect, and mammalian origin has become widespread. The popularity of this eukaryotic expression system is due to many factors, including (1) potentially high protein expression levels, (2) ease and speed of genetic engineering, (3) ability to accommodate large DNA inserts, (4) protein processing similar to higher eukaryotic cells (e.g., mammalia cells), and (5) ease of insect cell growth (e.g., suspension growth). The following review of the literature discusses two engineering aspects of recombinant protein synthesis by insect cell cultures: bioreactor scale-up and insect cell line selection. Following this review patent abstracts and additional literature pertaining to expression of recombinant proteins in insect cell culture are listed.     

The baculovirus display technology--an evolving instrument for molecular screening and drug delivery

High throughput screening is a core technology in drug discovery. During the past decade, several strategies have been developed to screen (poly)peptide libraries for diverse applications including disease diagnosis and profiling, imaging, as well as therapy. The recently established baculovirus display vector system (BDVS) represents a eukaryotic screening platform that combines the positive attributes of both cell and virus-based display approaches, allowing presentation of complex polypeptides on cellular and viral surfaces. Compared to microbial display systems, the BDVS has the advantage of correct protein folding and post-translational modifications similar to those in mammals, facilitating expression and analysis of proteins with therapeutic interest. The applicability of the system is further expanded by the availability of genetically engineered insect cell lines capable of performing e.g. mammalianized glycosylation in combination with high level of expression. In addition to insect cells, baculovirus can mediate delivery and expression of heterologous genes in a broad spectrum of primary and established mammalian cells. Currently, a variety of baculovirus-based assays aiming at routine high throughput identification of agents targeting cell surface receptors or studies on ligand-receptor interactions are under construction. Here, the advancements and future prospects of the baculovirus display technologies with emphasis on molecular screening and drug delivery applications using insect cell display, mammalian cell display, and virion display are described.     

Construction of the ie1-Bacmid Expression System and Its Use to Express EGFP and BmAGO2 in BmN Cells

The presently available expression tools and vectors (e.g., eukaryotic expression vectors and the adenovirus expression system) for studying the functional genes in Bombyx mori are insufficient. The baculovirus expression system is only used as a protein production tool; therefore, recombinant proteins expressed by B. mori using the baculovirus expression system equipped with a polyhedrin promoter cannot be used for in vivo research applications. In this work, we constructed and screened a eukaryotic expression vector for silkworm cells The EGFP and B. mori Argonaute2 proteins were found to be efficiently expressed using the screened pIEx-1 vector with the FuGENE 6 transfection reagent. Additionally, we constructed a novel nucleopolyhedrovirus ie1-Bacmid expression system for the production of recombinant protein; we then used the system to highly express the EGFP and B. mori Argonaute2 proteins. In this system, the protein of interest can be efficiently expressed 13 h after infection by controlling the B. mori nucleopolyhedrovirus immediate early ie1 promoter. The ie1-Bacmid system provides a powerful “adenovirus-like” expression tool; not only can the tool be used to study baculovirus molecular biology for the silkworm but it is also useful in other research applications as well, such as the study of gene functions involved in cellular physiological processes.     

Vector and Cell Line Engineering Technologies Toward Recombinant Protein Expression in Mammalian Cell Lines

The rapid growth of global biopharmaceutical market in the recent years has been a good indication of its significance in biotechnology industry. During a long period of time in recombinant protein production from 1980s, optimizations in both upstream and downstream processes were launched. In this regard, one of the most promising strategies is expression vector engineering technology based on incorporation of DNA opening elements found in the chromatin border regions of vectors as well as targeting gene integration. Along with these approaches, cell line engineering has revealed convenient outcomes in isolating high-producing clones. According to the fact that more than 50% of the approved therapeutic proteins is being manufactured in mammalian cell lines, in this review, we focus on several approaches and developments in vector and cell line engineering technologies in mammalian cell culture.     

Towards a Synthetic Biology Toolset for Metallocluster Enzymes in Biosynthetic Pathways: What We Know and What We Need

Microbes are routinely engineered to synthesize high-value chemicals from renewable materials through synthetic biology and metabolic engineering. Microbial biosynthesis often relies on expression of heterologous biosynthetic pathways, i.e., enzymes transplanted from foreign organisms. Metallocluster enzymes are one of the most ubiquitous family of enzymes involved in natural product biosynthesis and are of great biotechnological importance. However, the functional expression of recombinant metallocluster enzymes in live cells is often challenging and represents a major bottleneck. The activity of metallocluster enzymes requires essential supporting pathways, involved in protein maturation, electron supply, and/or enzyme stability. Proper function of these supporting pathways involves specific protein–protein interactions that remain poorly characterized and are often overlooked by traditional synthetic biology approaches. Consequently, engineering approaches that focus on enzymatic expression and carbon flux alone often overlook the particular needs of metallocluster enzymes. This review highlights the biotechnological relevance of metallocluster enzymes and discusses novel synthetic biology strategies to advance their industrial application, with a particular focus on iron-sulfur cluster enzymes. Strategies to enable functional heterologous expression and enhance recombinant metallocluster enzyme activity in industrial hosts include: (1) optimizing specific maturation pathways; (2) improving catalytic stability; and (3) enhancing electron transfer. In addition, we suggest future directions for developing microbial cell factories that rely on metallocluster enzyme catalysis.     

The baculovirus expression system as a tool for generating diversity by viral surface display

It has become a major goal of molecular biologists, biochemists, and immunologists to be able to modulate the structure of proteins, in order to increase their antigenicity, alter their biological properties and/or explore their function. Based on the concept of bacterial phage display, by which proteins are being selected and analyzed in conjunction with their genetic information, eukaryotic systems have been investigated for their use in generating biomolecular diversity. The advantage of posttranslational modification and the possible harbouring of structural complex proteins has lead scientists to include eukaryotic systems in the wide field of molecular design. The ideal expression vectors for surface display are eukaryotic viruses, that allow large gene insertions, efficiently present foreign proteins on the particle surface, are easy to propagate and, if possible, not pathogenic to humans. By inserting peptides into a native virus coat protein or by expressing foreign proteins as coat protein fusion proteins or linked to specific anchor domains it becomes possible to display polypeptides of interest on the surface of replicating particles. A variety of different strategies are currently under investigation in order to utilize the baculovirus insect cell expression system for efficient display on the surface of virus particles as well as on the surface of virally infected insect cells. Increasing the transfection efficiency, optimizing cloning procedures, and establishing applicable selection methods have lead to the development of a powerful tool for drug screening and ligand screening.     

Monitoring virus-like particle and viral protein production by intact cell MALDI-TOF mass spectrometry

A new application of intact cell MALDI-TOF MS (ICM-MS) methodology is described for monitoring the production of viral proteins and viral like particles using the baculovirus/insect cells expression system. Various MALDI matrices, cell preparation methods, cell/matrix volume ratio and MALDI target application procedures were tested in order to obtain the highest intensity and reproducibility of intact insect cell spectra. The web interface, SPECLUST (http://bioinfo.thep.lu.se/speclust.html), was used to construct dendograms based on MALDI-TOF MS data for evaluation of fingerprint changes.We demonstrate that insect cell mass spectrum fingerprints are characteristic of each viral protein/particle production. Their changes along the time for each production experiment correlate with the intracellular viral protein content determined by Western blot.This work shows that this simple, fast and low cost assay, which requires low sample volume, is a powerful analytical tool that complements the most common analytical methods used for monitoring bioprocesses and has potential application in the biotechnological industry namely, in the production of recombinant proteins.     

Cyclodextrins as Protective Agents of Protein Aggregation: An Overview

Cyclodextrins are extensively used in different fields (e.g., catalysis, chromatography, pharma, supramolecular chemistry, bioorganic chemistry, and bioinorganic chemistry), and their applications have been widely reviewed. Their main application in the field of pharmaceutical is as a drug carrier. This review overviews, for the first time, the use of cyclodextrins and their derivatives as antiaggregant agents in a number of proteins (e.g., amyloid‐β, insulin, recombinant human growth hormone, prion protein, transthyretin, and α‐synuclein) and some multimeric enzymes. There are many diseases that are correlated to protein misfolding and amyloid formation processes affecting numerous organs and tissues. There are over 30 different amyloid proteins and a number of corresponding diseases. Alzheimer's disease is the most common neurodegenerative disease. Treatment of these diseases is still a goal to reach, and many molecules are studied in this perspective. Cyclodextrins have also been studied, and they show great potential; as such, further studies could be very promising. This review aims to be a stimulus for the design of new cyclodextrin derivatives to obtain multifunctional systems with antiaggregant activity.     

Review applications of capillary electrophoresis to the analysis of biotechnology-derived therapeutic proteins

The number of proteins produced by recombinant DNA technology continues to grow at a rapid pace. In this review, the emphasis is on proteins that are of therapeutic interest. Aspects of protein analysis, such as glycoform separation of proteins produced in mammalian cells and the separation of oligosaccharides for structure elucidation, are covered. The use of antibodies as therapeutic proteins is growing and currently antibodies are the largest class of proteins produced by biotechnology. This has merited a separate section on analysis of antibodies by capillary electrophoresis (CE). Applications of mass spectrometry as an ancillary technique, used in conjunction with CE, are also covered briefly. This review covers the literature since 1999.     

Establishment of an ectopically expressed and functional PRMT1 for proteomic analysis of arginine-methylated proteins

Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), plays crucial roles in a variety of cellular processes. Mammalian PRMT1 exists in a large protein complex in cells, which has been implied in modulating the regulatory and catalytic properties of this enzyme. Establishment of a mammalian comparative approach will help to identify putative substrates of PRMT1 in an authentic condition. Here, we showed that ectopically expressed PRMT1 in mammalian HEK293 cells not only exhibited catalytic properties comparable to the endogenous enzyme but also existed in a functional complex together with endogenous PRMT1 and thus functioned as an endogenous counterpart. In addition, the measured methylation level of cellular proteins using a tritium-labeled methyl donor was accordingly enhanced upon ectopic expression of PRMT1. Subsequent proteomic analysis with such PRMT1-expressing cells allowed us to identify several known and putative methylated proteins. In vitro methylation of selected proteins, eukaryotic translation initiation factor 4A-I and vimentin, by cellular PRMT1 was shown. Together, we have demonstrated the functional equivalence of ectopically expressed PRMT1 in HEK293 cells and its application to systematically identify the substrate proteins in a mammalian cell context.     

Endothelial cell migration on surface-density gradients of fibronectin, VEGF, or both proteins

Cell migration is essential to many physiological processes, including angiogenesis, which is critical to the success of implanted biomaterials and tissue-engineered constructs. Gradients play an important role in cell migration. Previous work on cell migration has been mostly executed either in the concentration gradients of stimuli (e.g., VEGF) in bulk or hydrogels or on the surface-density gradients of ECM proteins (e.g., fibronectin) or small ligands (e.g., RGD). Little work has been done to investigate how cell migration responds to the surface-density gradients of growth factors. No work has been done to study how the surface gradients of both adhesive proteins and growth factors influence cell migration. In this work, we studied the effect of the surface-density gradients of fibronectin (FN), VEGF, or both proteins on endothelial cell migration. Gradients with different slopes were prepared to study how the gradient slope affects cell migration. The gradients were generated by first forming a counter-propagating C15COOH/C11OH self-assembled monolayer (SAM) gradient using a surface electrochemistry approach, followed by activating the -COOH moieties and covalently immobilizing proteins onto the surface. Fourier transform infrared spectra and X-ray photoelectron spectroscopy were used to characterize the SAM and protein gradients, respectively. A free cell migration assay using bovine aortic endothelial cells was performed on various gradient surfaces or on surfaces with uniform protein density. Results showed that cells on the surface-density gradients of FN, VEGF, or both proteins moved faster along the gradient direction than on the respective uniform control surface after 24-h cell culture. It is also shown that for each protein or protein combination, the directional cell displacement was not statistically different between two gradients with different slopes. Results show that the directional cell migration was increased by about 2-fold on the VEGF gradient as compared to the FN gradient and was further increased by another 2-fold on the combined gradients of both proteins as compared to the VEGF gradient alone. This is the first work to create surface-density gradients of VEGF and the first study to generate a combined surface gradient of growth factor and ECM protein to investigate their effect on cell migration on surfaces. This work broadens our understanding of the directional movement of endothelial cells. Our findings provide useful information for directing cell migration into tissue-engineered constructs and can be potentially used for those applications where cell migration is critical, such as angiogenesis.     

Differential display identifies genes in chinese hamster ovary cells sensitive to elevated ammonium

Ammonium is a toxic waste product that has been reported to negatively inhibit cell growth and recombinant glycosylation in Chinese hamster ovary (CHO) cells; however, the effect of this toxicity on intracellular gene expression has received only limited investigation. We used a differential display method to identify genes in CHO cells that were affected by ammonium stress. Eight genes whose mRNA levels significantly changed in response to elevated ammonium were isolated and identified. Five of the genes were identified as having lower expression under the ammonium stress, whereas three genes were identified as having higher expression. Sequence homology with other mammalian organisms was used to attribute function to these newly identified genes. The identified ammonium-sensitive genes were grouped into three broad functional groups: cellular processes, energy metabolism, and genetic-information processing. The three cellular process-related genes had lower expression (anaphase-promoting complex subunit 5, eukaryotic initiation factor 5A II, KIAA1091 protein). The two energy-related genes had higher expression under ammonium stress (adenosine triphosphate synthase subunit C and mitofusin 1). Both of the genetic information-processing genes (endoplasmic reticulum [ER]-resident protein ERdj5 and structure-specific recognition protein 1) had lower expression under the ammonium stress, whereas the 26S proteasome subunit adenosine triphosphatase 3 gene had higher expression. These preliminary results indicate that ammonium stress lowers expression of genes controlling cell cycle, protein folding, and quality and raises genes that control energy metabolism and degradation. Our findings demonstrate the usefulness of mRNA differential-display techniques for the detection of CHO cell genes affected by ammonium stress.     

Mass spectrometric analysis of protein phosphorylation

Phosphorylation is one of the most common posttranslational modifications of proteins in eukaryotic cells; it plays an important role in a wide spectrum of biological processes. This makes its study an important task for understanding cell functioning mechanisms. The aim of phosphoproteomics is a global mass spectral analysis of the phosphoprotein composition of cells, i.e., phosphoproteome. Nowadays, new effective methods are actively developed, which succeed not only in the detection of phosphorylated proteins but also in the determination of phosphorylated amino acid residues (phosphorylation sites) and in the quantitative comparison of phosphorylation among several specimens. Despite the analysis of protein phosphorylation remains a complicated problem, the available methods nowadays allow the detection of thousands of phosphorylation sites in the very same experiment. The present review covers the main methods utilized in contemporary phosphoproteomics: phosphoprotein and phosphopeptides enrichment as well as the mass spectrometric analysis of protein phosphorylation.     

Heterologous expression of photoactivated adenylyl cyclase (PAC) genes from the flagellate Euglena gracilis in insect cells

The unicellular, green flagellate wild-type Euglena gracilis (strain Z) possesses two genes of the photoactivated adenylyl cyclase (PAC) family. The corresponding gene products were found to be responsible for step-up (but not step-down) photophobic responses as well as both positive and negative phototaxis. The proteins consist of two PACalpha (Mr 105 kDa) and two PACbeta (90 kDa) subunits. In an effort to produce sufficient amounts of PAC proteins, several routes of over-expression have been tried including homologous expression in Euglena and heterologous expression in Escherichia coli. All these approaches were hampered by low yield or formation of inclusion bodies. Therefore we decided to attempt a heterologous expression in an insect cell line. PACalpha and PACbeta were separately cloned in the transfer vector pBacPAK9 with a His tag attached. The transfer vector was subsequently cotransfected via baculovirus into the insect cells and amplified. For the expression both recombinant viruses (containing PACbeta and PACbeta, respectively) were cotransfected simultaneously into insect cells. The expressed proteins were analyzed in Western blots using PACalpha and PACbeta antibodies. Most of the proteins were found to be in soluble form in high yield. The recombinant PAC proteins were purified via their attached His tag on an anti-His resin. Adenylyl cyclase activity was quantified after blue-light excitation using a cAMP enzyme immunoassay kit.     

Applications of capillary electrophoresis in biotechnology

Capillary electrophoresis (CE)-related techniques are increasingly being used as a matter of routine practice in the biotechnology discipline. Since recombinant DNA-derived proteins and the antisense oligonucleotides constitute a large portion of the applications of these techniques, they have been emphasized in this review. Analyses by CE of Escherichia coli-derived proteins and glycosylated proteins derived from mammalian cell cultures are summarized, as well as those of the carbohydrate chains that have been enzymatically removed from the protein. Applications of CE in the analysis of the antisense oligonucleotides for the determination of purity and the analytical studies on the metabolism of these modified oligonucleotides, by CE are reviewed. The literature mainly covers the period from 1996.     

Cloned s-Lap Gene Coding Area, Expression and Localization of s-Lap／GFP Fusion Protein in Mammal Cells

s-Lap is a new gene sequence from pig retinal pigment epithelial (RPE) cells, which was found and cloned in the early period of apoptosis of RPE cells damaged with visible light. We cloned the coding area sequence of the novel gene of s-Lap and constructed its recombinant eukaryotic plasmid pcDNA3.1-GFP/s-lap with the recombinant DNA technique. The expression and localization of s-lap/GFP fusion protein in CHO and B16 cell lines were studied with the instantaneously transfected pcDNA3.1-GFP/s-lap recombinant plasmid. s-Lap/GFP fusion protein can be expressed in CHO and B16 cells with a high rate expression in the nuclei.     

Design of Recombinant Stem Cell Factor–macrophage Colony Stimulating Factor Fusion Proteins and their Biological Activity <Emphasis Type="BoldItalic">In Vitro</Emphasis>

Summary Stem cell factor (SCF) and macrophage colony stimulating factor (M-CSF) can act in synergistic way to promote the growth of mononuclear phagocytes. SCF–M-CSF fusion proteins were designed on the computer using the Homology and Biopolymer modules of the software packages InsightII. Several existing crystal structures were used as templates to generate models of the complexes of receptor with fusion protein. The structure rationality of the fusion protein incorporated a series of flexible linker peptide was analyzed on InsightII system. Then, a suitable peptide GGGGSGGGGSGG was chosen for the fusion protein. Two recombinant SCF–M-CSF fusion proteins were generated by construction of a plasmid in which the coding regions of human SCF (1–165aa) and M-CSF (1–149aa) cDNA were connected by this linker peptide coding sequence followed by subsequent expression in insect cell. The results of Western blot and activity analysis showed that these two recombinant fusion proteins existed as a dimer with a molecular weight of ~84 KD under non-reducing conditions and a monomer of ~42 KD at reducing condition. The results of cell proliferation assays showed that each fusion protein induced a dose-dependent proliferative response. At equimolar concentration, SCF/M-CSF was about 20 times more potent than the standard monomeric SCF in stimulating TF-1 cell line growth, while M-CSF/SCF was 10 times of monomeric SCF. No activity difference of M-CSF/SCF or SCF/M-CSF to M-CSF (at same molar) was found in stimulating the HL-60 cell linear growth. The synergistic effect of SCF and M-CSF moieties in the fusion proteins was demonstrated by the result of clonogenic assay performed with human bone mononuclear, in which both SCF/M-CSF and M-CSF/SCF induced much higher number of CFU-M than equimolar amount of SCF or M-CSF or that of two cytokines mixture.     

Microscale control of cell contact and spacing via three-component surface patterning

The complexity of micropatterned cell constructs has been limited by difficulties in patterning more than two surface components on a culture substrate. Photolithography using multiple aligned masks is well established for generalized multicomponent patterning, but is often too harsh for biomolecules. We report a two-mask photolithographic process that is tuned to preserve bioactivity in patterns composed of covalently coupled poly(ethylene glycol) (PEG), adsorbed extracellular matrix protein (e.g., collagen I), and adsorbed serum proteins (e.g., vitronectin). Thereby, we pattern two cell types-primary hepatocytes and 3T3 fibroblasts-demonstrating control over contact and spacing (20-200 microm) between the two cell types for over one week. This method is applicable to the study of intercellular communication in cell biology and tissue engineering.     

Purification of Three Spectral Isoforms of Recombinant Fluorescent Protein from Insect Bioreactors Infected with Recombinant Baculovirus

Recombinant peptides and proteins have a wide range of applications, especially in medicine and biomedical research. Methods to rapidly and inexpensively manufacture recombinant proteins are needed to realize the full potential of proteins in medicine and to broaden their applications. A one-semester-length laboratory exercise is reported here demonstrating the use of the insect bioreactor system to create recombinant proteins. These laboratory exercises are suitable for students majoring in biochemistry or biotechnology and can be used to teach students basic biochemistry laboratory techniques including bacterial transformation, plasmid DNA isolation, insect cell culture, production of recombinant virus, use of recombinant viruses to deliver transgenes to a living organism, protein isolation by precipitation and chromatography, and protein quantification and protein characterization by electrophoresis and western blotting. The techniques acquired can also be used as the basis for related independent research projects in the second semester.