Baculovirus expression system for magnetic sorting of infected cells and enhanced titer determination

Recombinant baculoviruses derived from the Autographa californica nuclear polyhedrosis virus (AcNPV) are widely used to express heterologous genes in insect cells, but the use of the baculovirus expression vector system (BEVS) is hampered by slow and tedious procedures for the selection and separation of baculovirus-infected insect cells and for titer determination. Here we developed a new technology based on the bicistronic vector with a fusion protein of the human integral plasma membrane glycoprotein CD4 and green fluorescent protein (GFP) for concomitant expression of target proteins in insect Sf21 cells. Magnetic cell sorting (MACS) technology with anti-CD4 antibody-labeled superparamagnetic beads was used to separate the baculovirus-infected from the noninfected insect cells and therefore to increase the virus titer and to reduce process time. With the herein described use of the MACS-improved baculovirus expression plasmid MACS in baculovirus expression (pMACSiBac-1), we have been able to select the baculovirus-infected insect cells at an early time point of the infection cycle and therefore enrich the virus titer dramatically. Furthermore, simple end point dilution and GFP fluorescence detection can be used for early and facile detection of recombinant viruses and simplified titer determinations. We show that the bicistronic pMACSiBac-1 with an additional multiple cloning site under the control of the very late promoter polyhedrin (PPH) allows for the expression of target proteins in high amounts, less workloads, and shorter timelines.     

Cell-based analysis of Chikungunya virus E1 protein in membrane fusion

Background

Chikungunya fever is a pandemic disease caused by the mosquito-borne Chikungunya virus (CHIKV). E1 glycoprotein mediation of viral membrane fusion during CHIKV infection is a crucial step in the release of viral genome into the host cytoplasm for replication. How the E1 structure determines membrane fusion and whether other CHIKV structural proteins participate in E1 fusion activity remain largely unexplored.

Methods

A bicistronic baculovirus expression system to produce recombinant baculoviruses for cell-based assay was used. Sf21 insect cells infected by recombinant baculoviruses bearing wild type or single-amino-acid substitution of CHIKV E1 and EGFP (enhanced green fluorescence protein) were employed to investigate the roles of four E1 amino acid residues (G91, V178, A226, and H230) in membrane fusion activity.

Results

Western blot analysis revealed that the E1 expression level and surface features in wild type and mutant substituted cells were similar. However, cell fusion assay found that those cells infected by CHIKV E1-H230A mutant baculovirus showed little fusion activity, and those bearing CHIKV E1-G91D mutant completely lost the ability to induce cell-cell fusion. Cells infected by recombinant baculoviruses of CHIKV E1-A226V and E1-V178A mutants exhibited the same membrane fusion capability as wild type. Although the E1 expression level of cells bearing monomeric-E1-based constructs (expressing E1 only) was greater than that of cells bearing 26S-based constructs (expressing all structural proteins), the sizes of syncytial cells induced by infection of baculoviruses containing 26S-based constructs were larger than those from infections having monomeric-E1 constructs, suggesting that other viral structure proteins participate or regulate E1 fusion activity. Furthermore, membrane fusion in cells infected by baculovirus bearing the A226V mutation constructs exhibited increased cholesterol-dependences and lower pH thresholds. Cells bearing the V178A mutation exhibited a slight decrease in cholesterol-dependence and a higher-pH threshold for fusion.

Conclusions

Cells expressing amino acid substitutions of conserved protein E1 residues of E1-G91 and E1-H230 lost most of the CHIKV E1-mediated membrane fusion activity. Cells expressing mutations of less-conserved amino acids, E1-V178A and E1-A226V, retained membrane fusion activity to levels similar to those expressing wild type E1, but their fusion properties of pH threshold and cholesterol dependence were slightly altered.     

Stabilized baculovirus vector expressing a heterologous gene and GP64 from a single bicistronic transcript

The efficient scale-up of recombinant protein production in insect-cell bioreactors using baculovirus expression vectors is hampered by reductions in yield with increasing viral passage, the so-called passage effect. This phenomenon is characterized by the generation and subsequent accumulation of defective interfering baculoviruses (DIs), which interfere with the replication of genomically intact virus. A novel baculovirus expression vector is presented equipped with a bicistronic expression cassette that allows the simultaneous expression of the recombinant gene (GFP, first cistron) and an essential baculovirus gene (GP64, second cistron) from a single messenger RNA (mRNA). The translation of GP64 is mediated by an internal ribosome entry site (IRES) element from Rhopalosiphum padi virus (RhPV) while the native GP64 gene is deleted. In this way, a dominant selection pressure is placed on the entire bicistronic mRNA and hence on the maintenance of the foreign gene. The bicistronic expression vector was superior to the control baculovirus vector in that GFP expression remained at much higher levels upon continued virus passage. The versatility of this stabilized vector was demonstrated by its ability to propagate in a number of cell lines including Sf21, Sf9 and High Five cells. This novel baculovirus vector is especially valuable for large-scale recombinant protein production in insect-cell bioreactors where the number of viral passages is high.     

Baculovirus expression vectors that incorporate the foreign protein into viral occlusion bodies

Current baculovirus expression systems typically produce soluble proteins that accumulate within the infected insect cell or are secreted into the growth medium. A system has now been developed for the incorporation of foreign proteins, along with the matrix protein, polyhedrin, into baculovirus occlusion bodies. Initial studies showed that a recombinant virus expressing a translational fusion between polyhedrin and GFP did not form occlusion bodies. However, a baculovirus coexpressing native polyhedrin and the polyhedrin-GFP fusion protein formed occlusion bodies that fluoresced under UV light, demonstrating that they included the polyhedrin-GFP fusion protein. This was confirmed by immunoblot analysis. Thus, incorporation of a foreign protein into occlusion bodies depends on an interaction between native polyhedrin and the polyhedrin fusion protein. Electron microscopy demonstrated that the occlusion bodies containing GFP also incorporated virions as expected. These ColorPol occlusion bodies were as infectious to insect larvae as occlusion bodies produced by wild-type virus. This new system expands the capabilities for foreign gene expression by baculoviruses, which has implications for biopesticide design, novel vaccine delivery systems, and fusion protein purification applications.     

杆状病毒用于哺乳动物细胞快速高效表达外源基因的研究

现已发现杆状病毒可进入某些培养的哺乳动物细胞,这提示可将杆状病毒作为一种对哺乳动物细胞的新型基因转移载体。对杆状病毒转移载体的改造及对哺乳动物细胞的基因转移方式进行了进一步的研究。以绿色荧光蛋白基因为报告基因,利用Bac-to-Bac系统构建了分别含有正向和反向CMV启动子表达盒的两种重组杆状病毒。可观察到CMV启动子在Sf9细胞中可启动报告基因的表达,但表达效率较低。用重组杆状病毒感染后Sf9细胞的培养上清直接与HepG2细胞作用,以流式细胞术检测基因转移效率及荧光表达强度,发现这两种病毒在相同的感染复数下对HepG2细胞具有相似的基因转移及表达效率。同时,利用流式细胞术进一步研究了直接使用重组杆状病毒感染4d后Sf9细胞的培养上清对哺乳动物细胞进行基因转移的方法。通过对HepG2细胞的实验结果显示,将带毒Sf9细胞培养上清(1.2×10⁷PFU/mL)用哺乳动物细胞培养基1倍稀释后,37℃下孵育靶细胞12h(moi=50),可达到较高的基因转移及表达效率,同时不会对细胞造成明显损伤。将重组杆状病毒与脂质体和逆转录病毒这两种系统对HepG2及CV1细胞的基因转移效率进行了比较,结果发现在同样未经浓缩等特殊处理的条件下重组杆状病毒对这两种细胞的基因转移效率是最高的。因此可以认为,经过适当改造后的Bac-to-Bac重组杆状病毒系统可作为一种对哺乳动物细胞简便高效的基因转移表达载体。     

A baculovirus superinfection system: efficient vehicle for gene transfer into Drosophila S2 cells

The baculovirus expression vector system is considered to be a safe, powerful, but cell-lytic heterologous protein expression system in insect cells. We show here that there is a new baculovirus system for efficient gene transfer and expression using the popular and genetically well-understood Drosophila S2 cells. The recombinant baculovirus was constructed to carry an enhanced green fluorescent protein under the control of polyhedrin promoter as a fluorescent selection marker in the Sf21 cell line. Recombinant baculoviruses were then used to transduce S2 cells with target gene expression cassettes containing a Drosophila heat shock protein 70, an actin 5C, or a metallothionein promoter. Nearly 100% of the S2 cells showed evidence of gene expression after infection. The time course for the optimal protein expression peaked at 24 to 36 h postinfection, which is significantly earlier than a polyhedrin-driven protein expression in Sf21 cells. Importantly, S2 cells did not appear to be lysed after infection, and the protein expression levels are comparable to those of proteins under the control of polyhedrin promoter in several lepidopteran cell lines. Most surprisingly, S2 cells permit repetitive infections of multiple baculoviruses over time. These findings clearly suggest that this baculovirus-S2 system may effect the efficient gene transfer and expression system of the well-characterized Drosophila S2 cells.     

High-density perfusion culture of insect cells with a biosep ultrasonic filter

The baculovirus/insect cell expression system has provided a vital tool to produce a high level of active proteins for many applications. We have developed a very high-density insect cell perfusion process with an ultrasonic filter as a cell retention device. The separation efficiency of the filter was studied under various operating conditions. A cell density of over 30 million cells/mL was achieved in a controlled perfusion bioreactor and cell viability remained greater than 90%. Sf9 cells from a high-density culture and a spinner culture were infected with two recombinant baculoviruses expressing genes for the production of human chitinase and monocyte-colony inhibition factor. The protein yield on a cell basis from infecting high-density Sf9 cells was the same as or higher than that from the spinner Sf9 culture. Virus production from the high-density culture was similar to that from the spinner culture. The results show that the ultrasonic filter did not affect insect cells' ability to support protein expression and virus production following infection with baculovirus. The potential applications of the high-density perfusion culture for large-scale protein expression from Sf9 cells are also highlighted.     

Recombinant protein expression in aDrosophila cell line: comparison with the baculovirus system

In this report, we compare two different expression systems: baculovirus/Sf9 and stable recombinantDrosophila Schneider 2 (S2) cell lines. The construction of a recombinant S2 cell line is simple and quick, and in batch fermentations the cells have a doubling time of 20 hours until reaching a plateau density of 20 million cells/ml. Protein expression is driven by theDrosophila Metallothionein promoter which is tightly regulated. When expressed in S2 cells, the extracellular domain of human VCAM, an adhesion molecule, is indistinguishable from the same protein produced by baculovirus-infected Sf9 cells. Additionally, we present data on the expression of a seven trans-membrane protein, the dopamine D4 receptor, which has been successfully expressed in both systems. The receptor integrates correctly in the S2 membrane, binds [³H]spiperone with high affinity and exhibits pharmacological characteristics identical to that of the receptor expressed in Sf9 and mammalian cells. The general implications for large scale production of recombinant proteins are discussed.     

Baculovirus titration method based on MOI values for optimizing recombinant protein expression of the anti‐cancer vaccine candidate GA733‐Fc using Sf9 insect cells

The baculovirus expression system has been considered as a highly efficient tool for the production of recombinant biopharmaceutical proteins. The recombinant antigenic glycoprotein GA733 is a cell surface protein that is strongly expressed in human colorectal cancer. Efficient virus titration should be established to achieve optimal multiplicity of infection (MOI) conditions, which are in turn essential for strong expression of the recombinant GA733 fused to the human immunoglobulin IgG Fc fragment (GA733‐Fc) in the baculovirus‐insect system. In the present study, the Sf9 cell line was transfected with plasmid DNA containing the GA733‐Fc expression cassette under the control of the baculovirus polyhedron promoter. MOI values (0.05, 0.1, 0.5, 1, and 3) were calculated based on both microscope observations and results of titration assay and then used to determine the optimum recombinant expression and harvested sample [cell culture media (CM) or cell lysate (CL)]. The pFastBac dual vector carrying the GA733‐Fc gene was constructed to express GA733‐Fc and used to generate recombinant baculoviruses. Western blotting results showed that recombinant protein expression was dependent on the MOI. In addition, CM and CL showed significant differences in protein synthesis and protein secretion capacities. Our findings suggested that our proposed titration method can be used for reliable calculation of MOI values, which significantly influence recombinant GA733‐Fc protein expression in the baculovirus‐insect cell system.     

Estrogen receptor beta yield from baculovirus lytic infection is higher than from stably transformed Sf21 cells

The production of estrogen receptors (ER) in cultured insect cells is advantageous because these cells are relatively easy to culture and they perform post-translation modifications necessary for protein stability and function. There are three options for protein expression in insect cells: transient transfection, lytic baculovirus infection, or transfection followed by selection to create stable cell lines. Stable transfection has been promoted to be advantageous for the production of recombinant proteins because no re-infection is required, which might provide better lot-to-lot reproducibility in protein production. In this paper, we demonstrate that lytic baculovirus infection of Sf21 cells yields approximately tenfold more bioactive ERβ than cells stably transformed with pIZ/V5-His plasmid under OpIE2 promoter. We provide the first evidence that stable expression of recombinant human ERβ decreases the proliferation of Sf21 cells by inhibition of cell replication in a ligand-independent manner. These results mirror findings in breast cancer cells showing that an increase in ERβ expression decreases cell proliferation. We conclude that baculovirus infection of Sf21 cells is better for human ERβ production than stable-transformation of Sf21 cells.     

利用昆虫杆状病毒表达SARS冠状病毒的刺突蛋白和膜蛋白

SARS冠状病毒是人的严重急性呼吸综合征的病原体。对其他种类冠状病毒的研究结果显示,刺突蛋白(S蛋白)和膜蛋白(M蛋白)是病毒主要的结构蛋白。重组M蛋白和S蛋白可被用来作为抗原检测冠状病毒的感染和制备疫苗。这两个蛋白质分别被克隆并重组到昆虫杆状病毒基因组中,利用重组杆状病毒感染昆虫细胞来表达重组M蛋白和S蛋白,并对M蛋白进行了细胞内定位,融合蛋白的绿色荧光暗示了该蛋白质定位在细胞膜上。     

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.     

Expression of double foreign protein types following recombinant baculovirus infection of stably transfected Drosophila S2 cells

We sought to develop a platform for simultaneous, regulatable expression of double foreign protein types in cell culture. Drosophila melanogaster Schneider line 2 (S2) insect cells that stably express human erythropoietin (hEPO) were infected with a recombinant baculovirus containing the green fluorescent protein (GFP) gene. Since baculovirus cannot replicate in nonpermissive S2 cells, baculovirus infection did not affect cell growth or viability. Expression of each foreign protein was under the control of the inducible metallothionein (MT) promoter. Addition of copper sulfate to infected, stably transfected cells resulted in simultaneous expression of both GFP and hEPO. Induced hEPO expression profile and levels were similar in both control and infected cells, indicating that baculovirus infection also did not affect expression of stably introduced foreign gene. GFP protein levels were regulated by the infection dose of recombinant baculovirus, while hEPO expression remained constant. hEPO levels were much higher (30-fold) than GFP, indicating plasmid-based introduced gene copies have higher expression than baculovirus-based introduced genes. These data suggest the baculovirus/stable S2 cell system can be used to produce a major target protein by plasmid-based stable transfection, and assistant proteins by recombinant baculovirus infection. Such a system appears to be very attractive as a multiple protein expression platform for engineering metabolic pathways in cell culture.     

HCV核心蛋白在昆虫细胞中的表达及其免疫学特性的初步评价

通过逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）从丙肝患者的血清中分离出编码完整ＨＣＶ核心蛋白（Ｃ区）的ｃＤＮＡ片段,并将其克隆到杆状病毒转移质粒中。重组转移质粒ＤＮＡ与线性的杆状病毒ＤＮＡ共转染Ｓｆ９昆虫细胞,经蚀斑筛选获得了带编码全部核心蛋白基因的重组杆状病毒。重组病毒感染细胞后表达ＨＣＶ核心蛋白,其分子量的为２０ｋＤ。免疫印染和酶联免疫实验表明,此重组蛋白能被人ＨＣＶ阳性血清所识别。动物实验表明此重组蛋白能诱导小鼠产生特异性抗体。     

A strategy for generating monoclonal antibodies against recombinant baculovirus-produced proteins: application to the Bcl-2 oncoprotein.

A strategy is described for production of monoclonal antibodies against recombinant proteins that are produced using the baculovirus expression system and that requires no prior purification of the protein of interest. Crude lysates prepared from cultured Sf9 insect cells infected with recombinant or control baculoviruses are absorbed to nitrocellulose filters and used in a dot-immunobinding assay for screening hybridomas. The monoclonal antibody-producing hybridomas are derived by immunization of mice with a synthetic peptide corresponding to a hydrophilic region in the recombinant protein of interest. By using the baculovirus-produced recombinant protein as the screening antigen and by comparing antibody binding to filters containing control Sf9 lysates, hybridomas are identified that produce monoclonal antibodies with specific reactivity for the recombinant protein of interest and that can then subsequently be used to assist in the large-scale purification of the recombinant protein from baculovirus-infected cells. We applied this method to recombinant 26-kDa human Bcl-2 (B-cell lymphoma/leukemia-2), an integral membrane oncoprotein that regulates programmed cell death ("apoptosis") in hematolymphoid cells through unknown mechanisms. Two mouse monoclonal antibodies were produced that specifically bound the recombinant Bcl-2 baculoprotein in both solution and solid-phase assays.     

Expression of three reporter genes in four cell lines developed from <Emphasis Type="Italic">Papilio demoleus</Emphasis> Linnaeus (Lepidoptera: Papilionidae)

This paper used recombinant baculoviruses that carried three reporter genes, green fluorescent protein (GFP), β-galactosidase, and secreted alkaline phosphatase (SEAP), to infect four new cell lines from Papilio demoleus Linnaeus larvae (named RIRI-PaDe-1, RIRI-PaDe-2, RIRI-PaDe-3, and RIRI-PaDe-4). The expression levels of the three recombinant proteins were detected at 24, 48, 72, 96, 120, and 144 h after infection and compared with Sf9 and High Five cells to evaluate the characteristics of these four cell lines as host cells. The inoculation densities of the tested cell lines were 2?×?10⁴ cells/well (96-well plate) and 1?×?10⁵ cells/well (24-well plate), and adding a volume of virus stock resulted in an MOI of 5.0. The results showed that the four cell lines could be infected by recombinant baculovirus and that cell lysis occurred 96 h after infection. In the four tested cell lines, only a small number of RIRI-PaDe-1 and RIRI-PaDe-3 cells expressed recombinant GFP and showed green fluorescence. The expression was much lower than that of Sf9 and High Five. Comparing the intracellular and extracellular activity of β-galactosidase indicated that the P. demoleus cell system was more suitable for the expression of secreted proteins, and its extracellular β-galactosidase level was close to that of Sf9, but the expression level of SEAP was far lower than those of Sf9 and High Five.     

Optimization of colorectal cancer vaccine candidate protein GA733‐Fc expression in a baculovirus–insect cell system

The baculovirus–insect cell expression system has been used to produce functional recombinant proteins. The antigen GA733 is a cell‐surface glycoprotein highly expressed on most human colorectal carcinoma cells. Conditions for the expression of GA733 fused to the human immunoglobulin IgG Fc fragment (GA733‐Fc) were optimized in the baculovirus expression system. Several variable factors were adjusted to optimize expression, including the cell line (Sf9 and High Five), multiplicity of infection (MOI) value (0.05, 0.1, 0.5, 1 and 3), post‐infection time (48, 72 and 96 h) and harvested sample (cell culture media (CM) or cell lysate (CL)). In addition, two pFastBac Dual vectors carrying the GA733‐Fc gene were constructed to express GA733‐Fc with or without an endoplasmic reticulum (ER) retention sequence KDEL and used to generate recombinant baculoviruses. Western blot showed that expression depended on the conditions used to express the recombinant proteins. The protein production level and secretion capability differed in each cell line. In Sf9 cells, the highest expression in the CM and CL was obtained with GA733‐Fc at 96 h post‐infection at 0.1 MOI and with GA733‐FcK at 96 h post‐infection at 3 MOI, respectively. In High Five cells, the highest expression in the CM and CL was obtained with GA733‐Fc at 48 h post‐infection at 1 MOI and with GA733‐FcK at 48 h post‐infection at 3 MOI, respectively. These results suggest that the MOI value, post‐infection time and subcellular localization affect expression, and that these conditions can be modified to optimize protein expression in the baculovirus–insect cell system.     

Establishment of insect cell lines expressing green fluorescent protein on cell surface based on AcMNPV GP64 membrane fusion characteristic

Displaying a protein on the surface of cells has been provided a very successful strategy to function research of exogenous proteins. Based on the membrane fusion characteristic of Autographa californica multiple nucleopolyhedrovirus envelope protein GP64, we amplified and cloned N-terminal signal peptide and C-terminal transmembrane domain as well as cytoplasmic tail domain of gp64 gene into vector pIZ/V5-His with multi-cloning sites to construct the cell surface expression vector pIZ/V5-gp64. To verify that the vector can be used to express proteins on the membrane of insect cells, a recombinant plasmid pIZ/V5-gp64-GFP was constructed by introducing the PCR amplified green fluorescent protein (GFP) gene and transfected into insect cell lines Sf9 and H5. The transected cells were screened with zeocin and cell cloning. PCR verification results showed that the GFP gene was successfully integrated into these cells. Green fluorescence in Sf9-GFP and H5-GFP cells was observed by using confocal laser scanning microscopy and immunofluorescence detection indicated that GFP protein was located on the cell membrane. Western blot results showed that a fusion protein GP64-GFP of about 40 kDa was expressed on the membrane of Sf9-GFP and H5-GFP cells. The expression system constructed in this paper can be used for localization and continuous expression of exogenous proteins on insect cell membrane.     

Time reduction and process optimization of the baculovirus expression system for more efficient recombinant protein production in insect cells

Rapid expression of recombinant proteins for structure determination is one of the major challenges in pharmaceutical and academic research, since the number of potential drug targets has increased significantly in the last decade. Despite the fact that the baculovirus expression vector system is widely used for this purpose, the system is hampered by three very slow and tedious procedures, namely generation of high titer baculovirus stock, determination of the virus titer and discovery of the best conditions for protein expression. We herein describe the development of the ultraBac system to address and overcome these issues for protein expression in insect cells. We have established a new baculovirus expression technology for insect cells that is based on co-expression of GFP with target genes, a new regime for cell culturing and a highly efficient purification and enrichment procedure for recombinant baculovirus particles. Co-expression of GFP is used to monitor the infection of insect cells, to simplify titer determination and to optimize expression conditions. The new regime for cell culturing with increased viability of non-infected insect cells and its combination with the massive enrichment of virus particles via high-speed centrifugation enables the production of large amounts of recombinant virus in a very short period of time. By combining these techniques and by using the bicistronic vector pUltraBac-1, we have been able to cut the time-lines for protein expression in insect cells by half, approaching those for protein production in Escherichia coli. This new expression system is a significant step forward towards industrialized protein production in both, industry and academia.     

Functional expression of mammalian receptors and membrane channels in different cells

In native tissues, the majority of medically important membrane proteins is only present at low concentrations, making their overexpression in recombinant systems a prerequisite for structural studies. Here, we explore the commonly used eukaryotic expression systems-yeast, baculovirus/insect cells (Sf9) and Semliki Forest Virus (SFV)/mammalian cells-for the expression of seven different eukaryotic membrane proteins from a variety of protein families. The expression levels, quality, biological activity, localization and solubility of all expressed proteins are compared in order to identify the advantages of one system over the other. SFV-transfected mammalian cell lines provide the closest to native environment for the expression of mammalian membrane proteins, and they exhibited the best overall performance. But depending on the protein, baculovirus-infected Sf9 cells performed almost as well as mammalian cells. The lowest expression levels for the proteins tested here were obtained in yeast.