High production of heterologous proteins in Escherichia coli using the thermo-regulated T7 expression system

The exclusive use of isopropyl beta-D-thiogalactopyranoside to activate the T7 promoter for protein production has limited the general use of the expression system. We have sought an alternative by constructing a recombinant Escherichia coli strain, BL21 (G2), to carry a chromosomal copy of T7 gene 1 fused to the lambdaPL and lambdaP(R) tandem promoter. As a result, the recombinant strain harboring the carbamoylase gene from Agrobacterium radiobacter NRRL B11291 was shown to display various levels of.protein production in response to different degrees of heat shock. In particular, the system remained inactive at 30 degrees C and exhibited high sensitivity to heat such that a detectable carbamoylase activity could be measured after exposure to 33 degrees C. Moreover, heating in two steps - elevating the temperature from 30 degrees C to 39 degrees C and holding for a brief period, followed by reducing to 37 degrees C--was found to be the most potent method for protein production in this case. Using this approach, the recombinant protein accounted for 20% of total protein content of the cell. These results reveal the advantages of this expression system: responsiveness to thermal modulation and high-level production capability. In an attempt to enhance the total protein yield, a fed-batch fermentation process was carried out to control the cell growth rate by adjusting the substrate inflow. By applying the two-step temperature change. a carbamoylase yield with enzyme activity corresponding to 14,256 units was obtained. This production yield is a 10-fold increase in comparison with that at the batch-fermentation scale and 2,000-fold higher than that achieved at the shake-flask scale. Overall, it illustrates the promise of the newly constructed T7 system based on heat inducibility for industrial scale production of recombinant proteins.     

Employing Rhodobacter sphaeroides to functionally express and purify human G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the largest class of cell surface receptors and play crucial roles in many cellular and physiological processes. Functional production of recombinant GPCRs is one of the main bottlenecks to obtaining structural information. Here, we report the use of a novel bacterial expression system based on the photosynthetic bacterium Rhodobacter sphaeroides for the production of human recombinant GPCRs. The advantage of employing R. sphaeroides as a host lies in the fact that it provides much more membrane surface per cell compared to other typical expression hosts. The system was tailored to overexpress recombinant receptors under the control of the moderately strong and highly regulated superoperonic photosynthetic promoter pufQ. We tested this system for the expression of some class A GPCRs, namely, the human adenosine A2a receptor (A2aR), the human angiotensin AT1a receptor (AT1aR) and the human bradykinin B2 receptor (B2R). Several different constructs were examined and functional production of the recombinant receptors was achieved. The best-expressed receptor, AT1aR, was solubilized and affinity-purified. To the best of our knowledge, this is the first report of successful use of a bacterial host--R. sphaeroides--to produce functional recombinant GPCRs under the control of a photosynthetic gene promoter.     

A spontaneous runaway vector for production-scale expression of bovine somatotropin from Escherichia coli

An Escherichia coli expression vector was constructed for the production-scale fermentation of recombinant bovine somatotropin (rBST). Gene expression is regulated by a spontaneous increase in copy number at a constant low temperature without the need for an external inducer. This vector, designated pURA-4, contains the ampicillin resistance gene, the replication origin from pBR322, the R1 temperature-inducible runaway replicon, and a gene encoding rBST. Optimized rBST expression levels of >35% total cell protein were achieved at a constant 28 degrees C. Shake-flask analysis of pURA-4 shows that the copy number spontaneously increases approximately 6-fold during rBST production. Investigation into the mechanism of pURA-4 spontaneous runaway shows that the increase in copy number is directed by the pBR322 ori and not by the R1 replicon. Although the R1 temperature-inducible replicon does not mediate spontaneous runaway, it does have a positive effect on rBST expression. Copy number analysis also confirmed the stability of pURA-4 spontaneous runaway from the shake-flask scale through the production scale.     

The Development of a Novel Mycobacterium-Escherichia coli Shuttle Vector System Using pMyong2, a Linear Plasmid from Mycobacterium yongonense DSM 45126T

The Mycobacterium-Escherichia coli shuttle vector system, equipped with the pAL5000 replicon, is widely used for heterologous gene expression and gene delivery in mycobacteria. Despite its extensive use, this system has certain limitations, which has led to the development of alternative mycobacterial vector systems. The present study describes the molecular structure and expression profiles of a novel 18-kb linear plasmid, pMyong2, from Mycobacterium yongonense. Sixteen open reading frames and a putative origin of replication were identified, and the compatibility of the pMyong2 and pAL5000 vector systems was demonstrated. In recombinant Mycobacterium smegmatis (rSmeg), the pMyong2 vector system showed a copy number that was approximately 37 times greater than that of pAL5000. Furthermore, pMyong2 increased the mRNA and protein expression of the human macrophage migration inhibitory factor (hMIF) over pAL5000 levels by approximately 10-fold and 50-fold, respectively, demonstrating the potential utility of the pMyong2 vector system in heterologous gene expression in mycobacteria. Successful delivery of the EGFP gene into mammalian cells via rSmeg carrying the pMyong2 vector system was also observed, demonstrating the feasibility of this system for DNA delivery. In conclusion, the pMyong2 vector system could be effectively used not only for the in vivo delivery of recombinant protein and DNA but also for mycobacterial genetic studies as an alternative or a complement to the pAL5000 vector system.     

Investigation of the instability of plasmids directing the expression of Met-prochymosin in Escherichia coli

The causes of the instability of a multicopy plasmid, pCT70, which directs the expression of calf prochymosin in Escherichia coli, were investigated. Plasmid pAT153 and its derivative, pCT54, were stable for more than 90 generations in continuous culture with glucose limitation. The multicopy plasmid pCT66, which expressed very low levels of prochymosin due to poor translational efficiency, and low copy number plasmids which efficiently expressed the prochymosin gene, were also stable. These results indicated that high level translation of the recombinant gene was the cause of the instability of pCT70. The maximum specific growth rate of E. coli(pCT70) was reduced by 30% compared with E. coli(pCT66). To fulfil the requirements of a production system, a dual origin plasmid with controllable copy number was developed. Both this plasmid (pMG165) and a derivative which contained the prochymosin gene (pMG168) were stable when maintained at low copy number. When the copy number of plasmid pMG168 was increased by putting replication under the control of the lambda PR promoter and the cI857 temperature sensitive repressor, expression of prochymosin was achieved. This strategy enables large-scale production of prochymosin without the need for antibiotic selection or other methods of preventing plasmid loss.     

Stringent regulation and high-level expression of heterologous genes in Escherichia coli using T7 system controllable by the araBAD promoter

The recombinant Eschreichia coli strain BL21 (BAD) was constructed to carry a chromosomal copy of T7 gene 1 fused to the araBAD promoter. To further characterize this expression system, strain BL21 (BAD) was transformed with the plasmid containing the carbamoylase gene from Agrobacterium radiobacter driven by the T7 promoter. Upon induction with L-arabinose, recombinant cells produced 100-fold increase in carbamoylase activity in comparison with uninduced cells on M9 semidefined medium plus glycerol. This protein yield accounts for 30% of total cell protein content. In addition, it was found that after 100 generations the plasmid harboring the carbamoylase gene remained firmly stable in strain BL21 (BAD), but its stability dropped to only 20-30% in strain BL21 (DE3), a commercial strain bearing T7 gene 1 regulated by the lacUV5 promoter in its chromosome. In an attempt to enhance the total protein yield, fed-batch fermentation process was carried out using a two-stage feeding strategy to compartmentalize cell growth and protein synthesis. In the batch fermentation stage, the culture was grown on glucose to reach the stationary growth phase. Subsequently, glycerol was fed to the culture broth and L-arabinose was augmented to induce protein production when cells entered the late log growth phase. As a result, a carbamoylase yield corresponding to 5525 units was obtained, which amounts to a 337-fold increase over that achieved on a shake-flask scale. Taken together, these results illustrate the practical usefulness of T7 system under control of the araBAD promoter for heterologous protein production.     

High level expression of biologically active estrogen receptor in Saccharomyces cerevisiae

Biochemical over-expression of the human estrogen receptor was achieved using a Saccharomyces cerevisiae expression system. The receptor was produced as a novel ubiquitin fusion protein. This fusion protein is short lived in the cell and is processed to produce unfused receptor shortly after folding. Conventional high copy expression plasmids produced receptor to about 0.04% of the total soluble protein. By incorporating a defective leu2 allele into these vectors, an additional 5-fold increase in receptor production was obtained. The recombinant receptor was undergraded, soluble and biologically active. Conventional methods of disrupting cells using glass beads had a detrimental effect on the ability of the receptor to bind hormone. Enzymatic digestion of the cell wall followed by hypotonic shock liberates the receptor that quantitatively binds estrogen.     

Evolved Lactococcus lactis Strains for Enhanced Expression of Recombinant Membrane Proteins

The production of complex multidomain (membrane) proteins is a major hurdle in structural genomics and a generic approach for optimizing membrane protein expression is still lacking. We have devised a selection method to isolate mutant strains with improved functional expression of recombinant membrane proteins. By fusing green fluorescent protein and an erythromycin resistance marker (ErmC) to the C-terminus of a target protein, one simultaneously selects for variants with enhanced expression (increased erythromycin resistance) and correct folding (green fluorescent protein fluorescence). Three evolved hosts, displaying 2- to 8-fold increased expression of a plethora of proteins, were fully sequenced and shown to carry single-site mutations in the nisK gene. NisK is the sensor protein of a two-component regulatory system that directs nisin-A-mediated expression. The levels of recombinant membrane proteins were increased in the evolved strains, and in some cases their folding states were improved. The generality and simplicity of our approach allow rapid improvements of protein production yields by directed evolution in a high-throughput way.     

增加植酸酶基因appA-m的拷贝提高其在巴斯德毕赤酵母的表达量

为了进一步提高植酸酶的发酵效价,降低植酸酶生产成本,对毕赤酵母表达载体pGAPZα-A进行了改造。将表达载体pPIC9的AOX1启动子序列引入pGAPZα-A,使之成为甲醇可诱导型表达载体pAOXZα,插入植酸酶基因appA-m后得到重组载体pAOXZα-appA-m。以染色体上带有一个拷贝的appA-m基因、发酵效价可达到7.5×106IU/mL发酵液的重组酵母菌株74#为受体菌进行转化,在该重组菌株的染色体上的另一位点整合含有植酸酶基因的表达盒,经筛选到高表达植酸酶的重组子。通过PCR进行验证,植酸酶基因被整合到重组酵母的染色体上,且受体菌中原有的植酸酶基因结构未改变。重组菌在5L发酵罐经甲醇诱导120h植酸酶蛋白表达量达到4mg/mL发酵液,酶活性(发酵效价)达到1.2×107IU/mL发酵液以上,较含单拷贝植酸酶基因的受体菌株表达量有较大程度提高。PCR检测及表达量分析证明改良的菌株具有很好的遗传稳定性和表达稳定性。     

Cloning of heterologous genes specifying detrimental proteins on pUC-derived plasmids inEscherichia coli

A system is described that enables the cloning of genes specifying detrimental proteins inEscherichia coli. The system is based on pUC plasmids and was developed for the expression of theBacillus subtilis csaA gene, which is lethal when expressed at high levels. Suppressor strains that tolerate the presence of plasmids for high-level expression ofcsaA were isolated, which contained small cryptic deletion variants of the parental plasmid in high copy numbers. The cryptic plasmids consisted mainly of the pUC replication functions and lacked thecsaA region and selectable markers. The co-resident, incompatible, cryptic plasmids enabled the maintenance of thecsaA plasmids by reducing their copy number 20-fold, which resulted in a concomitant 3- to 7-fold reduction in the expression of plasmid-encoded genes. Strains carrying these cryptic endogenous plasmids proved to be useful for the construction of pUC-based recombinant plasmids carrying other genes, such as theskc gene ofStreptococcus equisimilis, which cannot be cloned in high copy numbers inE. coli. Several strategies to reduce production levels of heterologous proteins specified by plasmids are compared.     

Cloning of heterologous genes specifying detrimental proteins on pUC-derived plasmids inEscherichia coli

A system is described that enables the cloning of genes specifying detrimental proteins inEscherichia coli. The system is based on pUC plasmids and was developed for the expression of theBacillus subtilis csaA gene, which is lethal when expressed at high levels. Suppressor strains that tolerate the presence of plasmids for high-level expression ofcsaA were isolated, which contained small cryptic deletion variants of the parental plasmid in high copy numbers. The cryptic plasmids consisted mainly of the pUC replication functions and lacked thecsaA region and selectable markers. The co-resident, incompatible, cryptic plasmids enabled the maintenance of thecsaA plasmids by reducing their copy number 20-fold, which resulted in a concomitant 3- to 7-fold reduction in the expression of plasmid-encoded genes. Strains carrying these cryptic endogenous plasmids proved to be useful for the construction of pUC-based recombinant plasmids carrying other genes, such as theskc gene ofStreptococcus equisimilis, which cannot be cloned in high copy numbers inE. coli. Several strategies to reduce production levels of heterologous proteins specified by plasmids are compared.     

A Saccharomyces cerevisiae autoselection system for optimised recombinant protein expression

Yeasts are attractive organisms for recombinant protein production. They combine highly developed genetic systems and ease of use with reductions in time and costs. We describe an autoselection system for recombinant protein expression in Saccharomyces cerevisiae which increases yields 5-10-fold compared to conditional selection for expression plasmids. Multicopy expression plasmids encoding essential MOB1 or CDC28 genes are absolutely necessary for the viability of host cells with mob1 or cdc28 deletions in their genomes. Such plasmids are stably maintained, even in rich medium, so optimising biomass production and yields of recombinant protein. Plasmid copy numbers are also increased by limiting selective MOB1 and CDC28 gene expression prior to induction. GST- or 6His-tagged proteins are produced for affinity purification and are expressed from a conditional GAL1-10 promoter to avoid potentially toxic effects of recombinant proteins on growth. Autoselection systems for expressing single or pairs of proteins are described. We demonstrate the versatility of this system by expressing proteins from a number of organisms and include several large and problematic products. The in vitro reconstruction of a step in mitotic regulation shows how this expression system can be successfully applied to the detailed analysis of complex metabolic pathways.     

Optimizing functional versus total expression of the human mu-opioid receptor in Pichia pastoris

The expression of the EGFP-human mu-opioid receptor fusion protein in the methylotrophic yeast Pichia pastoris was optimized and monitored using both fluorescence and ligand-binding experiments. A set of parameters, including gene copy number, strain type, temperature, pH, and methanol inducer levels, was studied for its effect on the production of the recombinant protein. We show here that the expression level is optimal after 10 h of promoter induction and that the maximum is reached at a lower temperature and a higher pH than normally used. The optimized conditions have allowed a fourfold increase of the ligand-binding active form of the receptor, whereas the total expression level determined by EGFP fluorescence measurements was not modified.     

Coexpression of BiP increased antithrombotic hirudin production in recombinant Saccharomyces cerevisiae

In order to increase a production level of antithrombotic hirudin, BiP was simultaneously expressed in recombinant Saccharomyces cerevisiae strains carrying ten and 15 copies of the hirudin expression cassette integrated in the chromosome. Coexpression of BiP greatly enhanced both cell growth and hirudin production in recombinant S. cerevisiae. Maximum hirudin concentration of 36 mg l(-1) was obtained from batch culture of the ten copy-number transformant concomitantly harboring an episomal copy of the BiP gene under the control of the GAL1 promoter, which is corresponding to a 2.5-fold increase compared with the control strain carrying the genomic BiP gene only. The mean size of the recombinant yeast cells expressing the BiP gene remained at a relatively constant level compared with the control strains of which size increased after the onset of hirudin expression by the GAL10 promoter.     

A large‐scale expression strategy for multimeric extracellular protein complexes using Drosophila S2 cells and its application to the recombinant expression of heterodimeric ligand‐binding domains of taste receptor

Many of the extracellular proteins or extracellular domains of plasma membrane proteins exist or function as homo‐ or heteromeric multimer protein complexes. Successful recombinant production of such proteins is often achieved by co‐expression of the components using eukaryotic cells via the secretory pathway. Here we report a strategy addressing large‐scale expression of hetero‐multimeric extracellular domains of plasma membrane proteins and its application to the extracellular domains of a taste receptor. The target receptor consists of a heterodimer of T1r2 and T1r3 proteins, and their extracellular ligand binding domains (LBDs) are responsible for the perception of major taste substances. However, despite the functional importance, recombinant production of the heterodimeric proteins has so far been unsuccessful. We achieved the successful preparation of the heterodimeric LBD by use of Drosophila S2 cells, which have a high secretory capacity, and by the establishment of a stable high‐expression clone producing both subunits at a comparable level. The method overcame the problems encountered in the conventional transient expression of the receptor protein in insect cells using baculovirus or vector lipofection, which failed in the proper heterodimer production because of the biased expression of T1r3LBD over T1r2LBD. The large‐scale expression methodology reported here may serve as one of the considerable strategies for the preparation of multimeric extracellular protein complexes.     

Regulated expression of active biotinylated G-protein coupled receptors in mammalian cells

We have developed a mammalian expression system suitable for the production of enzymatically biotinylated integral membrane proteins. The key feature of this system is the doxycycline (dox)-regulated co-expression of a secreted variant of Escherichia coli biotin ligase (BirA) and a target protein with a 13-residue biotin acceptor peptide (BioTag) appended to its extracellular domain. Here we describe the expression and functional analysis of three G-protein coupled receptors (GPCRs): protease-activated receptors (PARs) 1 and 2, and the platelet ADP receptor, P2Y(12). Clonal Chinese hamster ovary (CHO) Tet-On cell lines that express biotinylated GPCRs were rapidly isolated by fluorescence-activated cell sorting following streptavidin-FITC staining, thereby circumventing the need for manual colony picking. Analysis by Western blotting with streptavidin-HRP following endoglycosidase treatment revealed that all three GPCRs undergo N-linked glycosylation. The expression of biotinylated GPCRs on the cell surface was regulated by the concentration of dox in the medium, reaching a maximum at approximately 1 microg/mL dox. Similarly, the extent of GPCR biotinylation was dependent on biotin concentration, with maximum and complete biotinylation achieved upon supplementation with 50 microM biotin. Biotinylated PAR1 and PAR2 were readily and specifically cleaved on the surface of intact cells by their cognate proteases, and were capable of transducing extracellular stimuli, resulting in the downstream phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Notably, P2Y(12) mediated agonist-induced ERK phosphorylation only when it was expressed at low levels on the cell surface, highlighting the utility of regulated expression for the production of functionally active GPCRs in mammalian cells.