Rapid expression screening of eukaryotic membrane proteins in Pichia pastoris

The overexpression of milligram quantities of protein remains a key bottleneck in membrane protein structural biology. A challenge of particular difficulty has been the overproduction of eukaryotic membrane proteins. In order to cope with the frequently poor expression levels associated with these challenging proteins, it is often necessary to screen a large number of homologues to find a well expressing clone. To facilitate this process using the heterologous, eukaryotic expression host Pichia pastoris, we have developed a simple fluorescent induction plate‐screening assay that allows for the rapid detection of well expressing clones of eukaryotic membrane proteins that have been fused to GFP. Using a eukaryotic membrane protein known to express well in P. pastoris (human aquaporin 4) and homologues of the ER associated membrane protein phosphatidylethanolamine N‐methyltransferase (PEMT), we demonstrate that when a large number of clones are screened, a small number of highly expressing “jackpot” clones can be isolated. A jackpot PEMT clone resulted in 5 mg/L yield after purification. The method allows for the facile simultaneous screening of hundreds of clones providing an alternate to in‐culture screening and will greatly accelerate the search for overexpressing eukaryotic membrane proteins.     

Optimization of tetracycline-responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells

The inducible T-REx system and other inducible expression systems have been developed in order to control the expression levels of recombinant protein in mammalian cells. In order to study the effects of heterologous protein expression on mammalian host behavior, the gene for recombinant Human transferrin (hTf) was integrated into HEK-293 cells and expressed under the control of the T-REx inducible technology (293-TetR-Hyg-hTf) or using a constitutive promoter (293-CMV-hTf). A number of inducible clones with variable expression levels were identified for the T-REx system with levels of hTf for the high expressing clones nearly double those obtained using the constitutive cytomegalovirus (CMV) promoter. The level of transferrin produced was found to increase proportionately with tetracycline concentration between 0 and 1 mug/mL with no significant increases in transferrin production above 1 mug/mL. As a result, the optimal induction time and tetracycline concentrations were determined to be the day of plating and 1 mug/mL, respectively. Interestingly, the cells induced to express transferrin, 293-TetR-Hyg-hTf, exhibited lower viable cell densities and percent viabilities than the uninduced cultures for multiple clonal isolates. In addition, the induction of transferrin expression was found to cause an increase in the expression of the ER-stress gene, BiP, that was not observed in the uninduced cells. However, both uninduced and induced cell lines containing the hTf gene exhibited longer survival in culture than the control cells, possibly as a result of the positive effects of hTf on cell survival. Taken together, these results suggest that the high level expression of complex proteins in mammalian cells can limit the viable cell densities of cells in culture as a result of cellular stresses caused by generating proteins that may be difficult to fold or are otherwise toxic to cells. The application of inducible systems such as the T-REx technology will allow us to optimize protein production while limiting the negative effects that result from these cellular stresses.     

Simplified high-throughput screening of AOX1-expressed laccase enzyme in Pichia pastoris

The heterologous protein expression in Pichia pastoris under the control of alcohol oxidase (AOX1)promoter comprises two steps, the growth and induction phases, which are time-consuming and technically demanding. Here, we describe an alternate method where expression is carried out directly in the methanol-containing medium. Using this method, we were successful in screening high-activity laccase clones from a library of laccase mutants generated by random mutagenesis. This simplified method not only saves time but also is highly efficient and can be used for screening a large number of clones.     

Production of recombinant proteins in <Emphasis Type="Italic">E. coli</Emphasis> by the heat inducible expression system based on the phage lambda pL and/or pR promoters

The temperature inducible expression system, based on the pL and/or pR phage lambda promoters regulated by the thermolabile cI857 repressor has been widely use to produce recombinant proteins in prokariotic cells. In this expression system, induction of heterologous protein is achieved by increasing the culture temperature, generally above 37°C. Concomitant to the overexpression of heterologous protein, the increase in temperature also causes a variety of complex stress responses. Many studies have reported the use of such temperature inducible expression system, however only few discuss the simultaneous stress effects caused by recombinant protein production and the up-shift in temperature. Understanding the integral effect of such responses should be useful to develop improved strategies for high yield protein production and recovery. Here, we describe the current status of the heat inducible expression system based on the pL and/or pR λ phage promoters, focusing on recent developments on expression vehicles, the stress responses at the molecular and physiological level that occur after heat induction, and bioprocessing factors that affect protein overexpression, including culture operation variables and induction strategies.     

Oxidative stress-resistance assay for screening yeast strains overproducing heterologous proteins

Many natural proteins have been developed into drugs and produced for direct application. Identifying improved hosts to achieve high-level heterologous protein production is a challenge in the study of heterologous protein expression in recombinant yeast. In this study, a novel high-throughput assay to screen such overproducing Saccharomyces cerevisiae strains was systematically developed. The protocol designed was based on screening host strain derivatives with increased superoxide dismutase dependent resistance to oxidative stress. Yeast cells transformed with recombinant plasmid carrying SOD1 gene as a reporter responded exquisitely to oxidative stress induced by elevated concentrations of paraquat. Improved yeast strains resulting from screening clones subjected to genome shuffling through selective pressure argue for a more effective screening system compared with traditonal selection. Moreover, this approach can be employed in general biochemical analysis without utilization of flow cytometry or well plate reader. Therefore, it is expected that the high-throughput assay would make superior strains producing heterologous proteins.     

Interplay of SOS induction,recombinant gene expression,and multimerization of plasmid vectors in Escherichia coli

Using pBR322- and pUC-derived plasmid vectors, a homologous (Escherichia coli native esterase) and three heterologous proteins (human interleukin-2, human interleukin-6, and Zymomonas levansucrase) were synthesized in E. coli IC2015(recA::lacZ) and GY4786 (sfiA::lacZ) strains. Via time-course measurement of beta-galactosidase activity in each recombinant culture, the SOS induction was estimated in detail and the results were systematically compared. In recombinant E. coli, the SOS response did not happen either with the recombinant insert-negative plasmid backbone alone or the expression vectors containing the homologous gene. Irrespective of gene expression level and toxic activity of synthesized foreign proteins, the SOS response was induced only when the heterologous genes were expressed using a particular plasmid vector, indicating strong dependence on the recombinant gene clone and the selection of a plasmid vector system. It is suggested that in recombinant E. coli the SOS response (i.e., activation of recA expression and initial sfiA expression) may be related neither to metabolic burden nor toxic cellular event(s) by synthesized heterologous protein, but may be provoked by foreign gene-specific interaction between a foreign gene and a plasmid vector. Unlike in E. coli XL1-blue(recA(-)) strains used, all expression vectors encoding each of the three heterologous proteins were multimerized in E. coli IC2015 strains in the course of cultivation, whereas the expression vectors containing the homologous gene never formed the plasmid multimers. The extent of multimerization was also dependent on a foreign gene insert in the expression vector. As a dominant effect of the SOS induction, recombinant plasmid vectors used for heterologous protein expression appear to significantly form various multimers in the recA(+) E. coli host.     

Transient transfection coupled to baculovirus infection for rapid protein expression screening in insect cells

Baculovirus infected insect cells are widely used for heterologous protein expression. Despite the power of this system, the use of baculovirus techniques for protein expression screening is hampered by the time and resources needed to generate each recombinant baculovirus. Here, we show that a transfection/infection based expression system is suitable for screening of expression constructs in insect cells and represents a valid alternative to other traditional screening methodologies using recombinant baculovirus. The described method is based on gene delivery by transfection coupled to the induction of protein expression by non-recombinant baculovirus infection. Vectors that control expression by a combination of the baculovirus promoters ie1 and p10 and the enhancer element hr5 are among the ones suitable for this method. Infection with non-recombinant baculovirus drastically increases the basal activity of these elements, leading to protein over-expression. Multiple vectors can be simultaneously co-transfected/infected, making transfection/infection amenable for screening of multiple co-expressed proteins and protein complexes. Taken together, our results prove that the transfection/infection protocol is a valid and innovative approach for increasing speed and reducing costs of protein expression screening for structural and functional studies.     

A Novel Methodology for Enhanced and Consistent Heterologous Expression of Unmodified Human Cytochrome P450 1B1 (CYP1B1)

Cytochrome P450 1B1 (CYP1B1) is a universal cancer marker and is implicated in many other disorders. Mutations in CYP1B1 are also associated with childhood blindness due to primary congenital glaucoma (PCG). To understand the CYP1B1 mediated etiopathology of PCG and pathomechanism of various cancers, it is important to carry out its functional studies. Heterologous expression of CYP1B1 in prokaryotes is imperative because bacteria yield a higher amount of heterologous proteins in lesser time and so the expressed protein is ideal for functional studies. In such expression system there is no interference by other eukaryotic proteins. But the story is not that simple as expression of heterologous CYP1B1 poses many technical difficulties. Investigators have employed various modifications/deletions of CYP N-terminus to improve CYP1B1 expression. However, the drawback of these studies is that it changes the original protein and, as a result, invalidates functional studies. The present study examines the role of various conditions and reagents in successful and consistent expression of sufficient quantities of unmodified/native human CYP1B1 in E. coli. We aimed at expressing CYP1B1 in various strains of E. coli and in the course developed a protocol that results in high expression of unmodified protein sufficient for functional/biophysical studies. We examined CYP1B1 expression with respect to different expression vectors, bacterial strains, types of culture media, time, Isopropyl β-D-1-thiogalactopyranoside concentrations, temperatures, rotations per minute, conditioning reagents and the efficacy of a newly described technique called double colony selection. We report a protocol that is simple, easy and can be carried out in any laboratory without the requirement of a fermentor. Though employed for CYP1B1 expression, this protocol can ideally be used to express any eukaryotic membrane protein.     

Cytochalasin D can improve heterologous protein productivity in adherent Chinese hamster ovary cells

We generated a series of adherent gene-amplified CHO clones expressing human secreted alkaline phosphatase (SEAP) as a model for heterologous protein production. Clones demonstrate a 26- to 52-fold increase in productivity compared to controls after dhfr/methotrexate-mediated gene amplification and clone selection. SEAP is stably expressed in these clones over at least a 6-week period without significant productivity loss. Two-dimensional protein electrophoresis identified 21 proteins that exhibited altered expression in clones of increasing SEAP productivity. Based on MALDI TOF/TOF mass spectrometry of relevant protein spots, changes in translation, energy pathways, chaperones, regulatory proteins, and cytoskeletal proteins were observed, including a 4-fold expression increase in actin capping protein. We hypothesized that an alteration of the actin cytoskeleton using cytochalasin D as a mimic for actin-capping protein could have a beneficial effect on heterologous protein secretion. Treatment with 0.5 mug/mL cytochalasin D increased SEAP productivity 2- to 3-fold compared to an amplified control which resulted in an increase in productivity from 52- to 150-fold compared to a nonamplified parent.     

Identification of the unfolded protein response (UPR)-related genes from Bombyx mori cell lines by a subtractive hybridization approach

The baculovirus expression vector system (BEVS) is one of the powerful insect cell systems for heterologous protein expression. However, over-expression of heterologous proteins in this system sometimes results in protein misfolding and aggregation because of insufficient levels of folding catalysts. In previous study using the differential screening (DS) method, we isolated only 40 differentially expressed genes after treatment with tunicamycin, an unfolded protein response (UPR) inducer. To isolate more protein folding catalysts from insect, we performed suppressive subtractive hybridization (SSH) with untreated and tunicamycin-treated Bm5 cell lines in this study. We could isolate 366 differentially expressed clones by SSH method and produced expressed sequence tags (ESTs). ESTs included the UPR pathway-related genes involved in protein folding, including heat shock proteins, molecular chaperones, foldases, as well as glycosylation and secretory pathway related genes. Identification of the tunicamycin responsive genes using SSH provides more information about the UPR-related genes in insect cells, and will facilitate modifications of the protein folding pathway in the ER to improve heterologous protein expression.     

Fluorescent labeling for clonal selection of Marc 145 cells secreting high levels of recombinant protein PBD-1

Despite the powerful impact gene expression markers like the green fluorescent protein (GFP) or enhanced GFP (EGFP) exert on linking the expression of recombinant protein for selection of high producers in recent years, there is still a strong incentive to develop more economical and efficient methods for isolating mammalian cell clones secreting high levels of recombinant proteins. Here we present a new method based on the co-expression of EGFP that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the related protein are linked by an internal ribosome entry site and thus are transcribed into the same mRNA in an independent translation process. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein in each clone. By expressing recombinant porcine β-defensin 1 in Marc 145 cells, we demonstrate the robustness and performance of this technique. The method can be served as an alternative to identify high-producer clones with various cell sorting methods.     

结核分枝杆菌膜蛋白的异源表达与纯化研究进展

膜蛋白是一类与生物膜相互作用、具有重要功能和独特结构的蛋白质。异源表达纯化一直是了解膜蛋白结构和功能的重要瓶颈。结核分枝杆菌作为典型的胞内致病菌,其膜蛋白的研究具有很好的代表性以及重要意义。目前用于表达膜蛋白的有大肠杆菌、酵母、哺乳动物细胞等表达系统,但结核菌膜蛋白的表达宿主还往往局限于大肠杆菌。异源表达需要综合考虑蛋白的来源、疏水性、跨膜区等特性。低温、加入共表达因子以及改变培养条件有助于结核菌膜蛋白的可溶性表达。另外,包涵体复性也是获得结核菌目的膜蛋白的重要途径。随着新的表达系统,新的促可溶表达策略,新的包涵体复性手段,新的纯化方法的应用,将有更多的膜蛋白异源表达纯化成功,为蛋白质功能研究奠定基础。     

LCR/MEL: a versatile system for high-level expression of heterologous proteins in erythroid cells.

We have used the human globin locus control region (LCR) to assemble an expression system capable of high-level, integration position-independent expression of heterologous genes and cDNAs in murine erythroleukaemia (MEL) cells. The cDNAs are inserted between the human beta-globin promoter and the second intron of the human beta-globin gene, and this expression cassette is then placed downstream of the LCR and transfected into MEL cells. The cDNAs are expressed at levels similar to those of the murine beta-globin in the induced MEL cells. Heterologous genomic sequences can also be expressed at similar levels when linked to to the LCR and beta-globin promoter. In addition we demonstrate that, after induction of differentiation, MEL cells are capable of secreting heterologous proteins over a prolonged time period, making this system suitable for use in continuous production systems such as hollow fibre bioreactors. The utility of the LCR/MEL cell system is demonstrated by the expression of growth hormone at high levels (greater than 100 mg/l) 7 days after induction. Since the expression levels seen do not depend upon gene amplification and are independent of the integration position of the expression cassette, it is possible to obtain clones with stable high-level expression within 3-4 weeks after transfection.     

High-throughput protein expression screening and purification in Escherichia coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, to obtain milligrams of soluble proteins is still challenging since many proteins are expressed in an insoluble form without optimization. Therefore when working with tens of proteins or protein domains it is recommended that high-throughput expression screening at a small scale (1-4ml of culture) is carried out to identify the optimal conditions for soluble protein production. Once determined, these culture conditions can be applied at a large scale to produce sufficient protein for structural or functional studies. We describe a procedure that has enabled the systematic screening of culture conditions or fusion-tags on hundreds of cultures per week. The analysis of the optimal conditions for the soluble production of these proteins helped us to design a simple and efficient protocol for soluble protein expression screening. This protocol has since been used on hundreds of proteins and is illustrated with the genome wide scale production of proteins containing the DNA binding domains of Ciona intestinalis.     

Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris

The expression of heterologous proteins may exert severe stress on the host cells at different levels. Depending on the specific features of the product, different steps may be rate-limiting. For the secretion of recombinant proteins from yeast cells, folding and disulfide bond formation were identified as rate-limiting in several cases and the induction of the chaperone BiP (binding protein) is described. During the development of Pichia pastoris strains secreting human trypsinogen, a severe limitation of the amount of secreted product was identified. Strains using either the AOX1 or the GAP promoter were compared at different gene copy numbers. With the constitutive GAP promoter, no effect on the expression level was observed, whereas with the inducible AOX1 promoter an increase of the copy number above two resulted in a decrease of expression. To identify whether part of the product remained in the cells, lysates were fractionated and significant amounts of the product were identified in the insoluble fraction containing the endoplasmic reticulum, while the soluble cytosolic fraction contained product only in clones using the GAP promoter. An increase of BiP was observed upon induction of expression, indicating that the intracellular product fraction exerts an unfolded protein response in the host cells. A strain using the GAP promoter was grown both on glucose and methanol and trypsinogen was identified in the insoluble fractions of both cultures, but only in the soluble fraction of the glucose grown cultures, indicating that the amounts and distribution of intracellularly retained product depends on the culture conditions, especially the carbon source.     

A GFP-based method facilitates clonal selection of transfected CHO cells

The identification of highly expressing clones is a crucial step in the development of cell lines for production of recombinant proteins. Here we present a method based on the co-expression of enhanced green fluorescent protein (EGFP) that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the protein of interest are linked by an internal ribosome entry site and thus are transcribed into the same mRNA but are translated independently. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein for each clone. By expressing recombinant growth factors in CHO cells, we demonstrate the robustness and performance of this technique. The method is an alternative to the identification of high-producer clones using various cell sorting methods, as it can be performed with standard laboratory equipment.     

A T7 RNA polymerase-dependent gene expression system for Bacillus megaterium

Gene expression systems based on the RNA polymerase of the bacteriophage T7 are often the ultimate choice for the high level production of recombinant proteins. During the last decade, the Gram-positive bacterium Bacillus megaterium was established as a useful host for the intra- and extracellular production of heterologous proteins. In this paper, we report on the development of a T7 RNA polymerase-dependent expression system for B. megaterium. The system was evaluated for cytosolic and secretory protein production with green fluorescent protein (GFP) from Aequoria victoria as intracellular and Lactobacillus reuteri levansucrase as extracellular model protein. GFP accumulated rapidly at high levels up to 50 mg/l shake flask culture intracellularly after induction of T7 RNA polymerase gene expression. The addition of rifampicin for the inhibition of B. megaterium RNA polymerase led to an increased stability of GFP. L. reuteri levansucrase was also successfully produced and secreted (up to 20 U/l) into the culture supernatant. However, parallel intracellular accumulation of the protein indicated limitations affiliated with the Sec-dependent protein translocation process.