Recombinant Hansenula polymorpha as a biocatalyst: coexpression of the spinach glycolate oxidase (GO) and the S. cerevisiae catalase T (CTT1) gene

 The methylotrophic yeast Hansenula polymorpha has been developed as an efficient production system for heterologous proteins. The system offers the possibility to cointegrate heterologous genes in anticipated fixed copy numbers into the chromosome. As a consequence coproduction of different proteins in stoichiometric ratios can be envisaged. This provides options to design this yeast as an industrial biocatalyst in procedures where several enzymes are required for the efficient conversion of a given inexpensive compound into a valuable product. To this end recombinant strains have been engineered with multiple copies of expression cassettes containing the glycolate oxidase (GO) gene from spinach and the catalase T (CTT1) gene from S. cerevisiae. The newly created strains produce high levels of the peroxisomal glycolate oxidase and the cytosolic catalase T. The strains efficiently convert glycolate into glyoxylic acid, oxidizing the added substrate and decomposing the peroxide formed during this reaction into water and oxygen. Received: 31 October 1995/Received last revision: 23 February 1996/Accepted: 4 March 1996     

Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications

Prokaryotic expression systems are superior in producing valuable recombinant proteins, enzymes and therapeutic products. Conventional microbial technology is evolving gradually and amalgamated with advanced technologies in order to give rise to improved processes for the production of metabolites, recombinant biopharmaceuticals and industrial enzymes. Recently, several novel approaches have been employed in a bacterial expression platform to improve recombinant protein expression. These approaches involve metabolic engineering, use of strong promoters, novel vector elements such as inducers and enhancers, protein tags, secretion signals, high-throughput devices for cloning and process screening as well as fermentation technologies. Advancement of the novel technologies in E. coli systems led to the production of “difficult to express” complex products including small peptides, antibody fragments, few proteins and full-length aglycosylated monoclonal antibodies in considerable large quantity. Wacker's secretion technologies, Pfenex system, inducers, cell-free systems, strain engineering for post-translational modification, such as disulfide bridging and bacterial N-glycosylation, are still under evaluation for the production of complex proteins and peptides in E. coli in an efficient manner.

This appraisal provides an impression of expression technologies developed in recent times for enhanced production of heterologous proteins in E. coli which are of foremost importance for diverse applications in microbiology and biopharmaceutical production.     

Expression of HBsAg and preS2-S protein in different yeast based system: A comparative analysis

We have expressed both S and preS2-S genes coding for the hepatitis B small (S) and medium (M) proteins, respectively, in different yeast based expression systems and compared the production level of the recombinant proteins. In Saccharomyces cerevisiae, viral genes were expressed under the inducible Gal10/cyc1 and the constitutive PGK promoters using 2 μ replicating vectors. We showed that the yield of S protein was higher than M protein under both inducible (14.27 vs 10.9 mg/l) and constitutive (9.18 vs 6.39 mg/l) conditions, respectively. In the methylotrophic yeast Pichia pastoris, the viral genes were expressed in GS115 (Mut⁺: Methanol Utilizing) and KM71 (Mut^S: Methanol Utilizing Slow) under the control of the alcohol oxidase promoter (AOX1). In Mut^S background, both S and preS2-S genes were expressed at higher levels than in Mut⁺. In attempt to increase the yield of recombinant viral proteins in S. cerevisiae, we have co-expressed both inducible and constitutive vectors harboring the S or preS2-S genes leading to recombinant strains called UTS (containing pDP/S + pYePIT/S) and UTP (containing pDP/preS2-S + pYePIT/preS2-S). We showed that the recombinant S and preS2-S proteins were successfully detected and the production level reached 18.31 mg/l for the S and 13.22 mg/l for the M proteins.Our comparative study provides evidence that in small scale, S. cerevisiae is more suitable for HBsAg and preS2-S proteins production than P. pastoris under inducible rather than constitutive condition.     

Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes

Background

Plants are increasingly being examined as alternative recombinant protein expression systems. Recombinant protein expression levels in plants from Tobacco mosaic virus (TMV)-based vectors are much higher than those possible from plant promoters. However the common TMV expression vectors are costly, and at times technically challenging, to work with. Therefore it was a goal to develop TMV expression vectors that express high levels of recombinant protein and are easier, more reliable, and more cost-effective to use.

Results

We have constructed a Cauliflower mosaic virus (CaMV) 35S promoter-driven TMV expression vector that can be delivered as a T-DNA to plant cells by Agrobacterium tumefaciens. Co-introduction (by agroinfiltration) of this T-DNA along with a 35S promoter driven gene for the RNA silencing suppressor P19, from Tomato bushy stunt virus (TBSV) resulted in essentially complete infection of the infiltrated plant tissue with the TMV vector by 4 days post infiltration (DPI). The TMV vector produced between 600 and 1200 micrograms of recombinant protein per gram of infiltrated tissue by 6 DPI. Similar levels of recombinant protein were detected in systemically infected plant tissue 10–14 DPI. These expression levels were 10 to 25 times higher than the most efficient 35S promoter driven transient expression systems described to date.

Conclusion

These modifications to the TMV-based expression vector system have made TMV vectors an easier, more reliable and more cost-effective way to produce recombinant proteins in plants. These improvements should facilitate the production of recombinant proteins in plants for both research and product development purposes. The vector should be especially useful in high-throughput experiments.     

CHLOROPLAST GENETIC TOOL FOR THE GREEN MICROALGAE HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE,VOLVOCALES)1

At present, there is strong commercial demand for recombinant proteins, such as antigens, antibodies, biopharmaceuticals, and industrial enzymes, which cannot be fulfilled by existing procedures. Thus, an intensive search for alternative models that may provide efficiency, safety, and quality control is being undertaken by a number of laboratories around the world. The chloroplast of the eukaryotic microalgae Haematococcus pluvialis Flotow has arisen as a candidate for a novel expression platform for recombinant protein production. However, there are important drawbacks that need to be resolved before it can become such a system. The most significant of these are chloroplast genome characterizations, and the development of chloroplast transformation vectors based upon specific endogenous promoters and on homologous targeting regions. In this study, we report the identification and characterization of endogenous chloroplast sequences for use as genetic tools for the construction of H. pluvialis specific expression vectors to efficiently transform the chloroplast of this microalga via microprojectile bombardment. As a consequence, H. pluvialis shows promise as a platform for expressing recombinant proteins for biotechnological applications, for instance, the development of oral vaccines for aquaculture.     

Protein A as a fusion partner for the expression of heterologous proteins in Lactobacillus

An expression system based on the Staphylococcus aureus protein A gene (spa) was developed to allow the production and export of proteins in Lactobacillus. Plasmid shuttle vectors were constructed that carried the eZZ gene, a synthetic gene based on the Protein A gene (spa) but lacking the carboxy-terminal membrane-anchoring region. A gene fusion was created between the eZZ gene and the VD4 region of a chlamydial major outer-membrane protein gene. Expression studies demonstrated the recognition of the spa regulatory signals by several Lactobacillus, with the recombinant protein being expressed (from 0.1 μg of EZZVD4 fusion protein per ml in L. plantarum up to 10 μg of EZZ protein per ml in L. fermentum) and exported (levels up to 20% in L. fermentum) in several Lactobacillus strains. Received: 27 August 1996 / Received revision: 27 November 1996 / Accepted: 29 November 1996     

Efficient secretory production of alkaline phosphatase by high cell density culture of recombinant Escherichia coli using the Bacillus sp. endoxylanase signal sequence

New secretion vectors containing the Bacillus sp. endoxylanase signal sequence were constructed for the secretory production of recombinant proteins in Escherichia coli. The E. coli alkaline phosphatase structural gene fused to the endoxylanase signal sequence was expressed from the trc promoter in various E. coli strains by induction with IPTG. Among those tested, E. coli HB101 showed the highest efficiency of secretion (up to 25.3% of total proteins). When cells were induced with 1 mM IPTG, most of the secreted alkaline phosphatase formed inclusion bodies in the periplasm. However, alkaline phosphatase could be produced as a soluble form without reduction of expression level by inducing with less (0.01 mM) IPTG, and greater than 90% of alkaline phosphatase could be recovered from the periplasm by the simple osmotic shock method. Fed-batch cultures were carried out to examine the possibility of secretory protein production at high cell density. Up to 5.2 g/l soluble alkaline phosphatase could be produced in the periplasm by the pH-stat fed-batch cultivation of E. coli HB101 harboring pTrcS1PhoA. These results demonstrate the possibility of efficient secretory production of recombinant proteins in E. coli by high cell density cultivation. Received: 8 September 1999 / Received revision: 3 January 2000 / Accepted 4 January 2000     

Industrial production of recombinant therapeutics in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its recent advancements

Nearly 30% of currently approved recombinant therapeutic proteins are produced in Escherichia coli. Due to its well-characterized genetics, rapid growth and high-yield production, E. coli has been a preferred choice and a workhorse for expression of non-glycosylated proteins in the biotech industry. There is a wealth of knowledge and comprehensive tools for E. coli systems, such as expression vectors, production strains, protein folding and fermentation technologies, that are well tailored for industrial applications. Advancement of the systems continues to meet the current industry needs, which are best illustrated by the recent drug approval of E. coli produced antibody fragments and Fc-fusion proteins by the FDA. Even more, recent progress in expression of complex proteins such as full-length aglycosylated antibodies, novel strain engineering, bacterial N-glycosylation and cell-free systems further suggests that complex proteins and humanized glycoproteins may be produced in E. coli in large quantities. This review summarizes the current technology used for commercial production of recombinant therapeutics in E. coli and recent advances that can potentially expand the use of this system toward more sophisticated protein therapeutics.     

Versatile Expression and Secretion Vectors for Bacillus subtilis

Most expression systems are based on Escherichia coli as the host strain because of the large availability of all kinds of vector plasmids. However, aside from the obvious advantages of E. coli systems, serious problems can occur during the process of heterologous gene expression and purification. Therefore, low expression rates, formation of inclusion bodies, improper protein-folding, and/or toxicity problems might enforce changing the expression host. Here we describe the construction of two new vectors, pBSMuL1 and pBSMuL2, for overexpression and secretion of heterologous proteins in Bacillus subtilis as an alternative expression host. The new plasmids combine several advantages in comparison to available Bacillus expression systems: an appropriate multiple cloning site consisting of 13 unique restriction sites, one (pBSMuL1) or two (pBSMuL2) strong constitutive promoters, a high efficient signal sequence for protein secretion, and the possibility to express proteins as His-tagged fusions for easy detection and purification. We have demonstrated the applicability of the novel vector plasmids for the production and purification of the heterologous cutinase from Fusarium solani pisi.     

Biosynthetic production of type II fish antifreeze protein: fermentation by Pichia pastoris

Sea raven type II antifreeze protein (SRAFP) is one of three different fish antifreeze proteins isolated to date. These proteins are known to bind to the surface of ice and inhibit its growth. To solve the three-dimensional structure of SRAFP, study its ice-binding mechanism, and as a basis for engineering these molecules, an efficient system for its biosynthetic production was developed. Several different expression systems have been tested including baculovirus, Escherichia coli and yeast. The latter, using the methylotrophic organism Pichia pastoris as the host, was the most productive. In shake-flask cultures the levels of SRAFP secreted from Pichia were up to 5 mg/l. The recombinant protein has an identical activity to SRAFP from sea raven serum. In order to increase yields further, four different strategies were tested in 10-l fermentation vessels, including: (1) optimization of pH and dissolved oxygen, (2) mixed feeding of methanol and glycerol with Mut^s clones, (3) supplementation of amino acid building blocks, and (4) methanol feeding with Mut⁺ clones. The mixed-feeding/Mut^s strategy proved to be the most efficient with SRAFP yields reaching 30 mg/l. Received: 19 November 1996 / Received revision: 29 January 1997 / Accepted: 7 March 1997     

Induction of an antibody response in mice against human papillomavirus (HPV) type 16 after immunization with HPV recombinant Salmonella strains

 Human papillomaviruses (HPV) are present in approximately 95% of all cervical carcinomas and the HPV E6 and E7 genes are continuously expressed in these lesions. There is also circumstantial evidence that often natural immunity against HPV is generated and that this is of influence on HPV-induced lesions. Stimulation of the immune system by proper presentation of relevant HPV antigens might, therefore, lead to a prophylactic or therapeutic immunological intervention for HPV-induced lesions. For this purpose we have expressed the E6 and E7 protein of HPV 16 in an attenuated strain of Salmonella typhimurium (SL3261, aroA mutation), which has been used extensively as a live vector. Live recombinant Salmonella vaccines have the ability to elicit humoral, secretory and cell-mediated immune responses, including cytotoxic T cells, against the heterologous antigens they express. This report describes the construction of recombinant Salmonella strains expressing the HPV 16 E6 and E7 proteins, and the induction of an HPV-16-specific immune response in mice after immunization with these live vectors. Received: 25 June 1996 / Accepted: 6 August 1996     

Overproduction of d-hydantoinase and carbamoylase in a soluble form in Escherichia coli

The production of d-hydantoinase and carbamoylase from Agrobacterium radiobacter NRRL B11291 using T7 and trc promoters, respectively, was found to cause protein aggregates in Escherichia coli. We initiated a systematic study aimed at overproducting these two proteins in a soluble form. As a result, the protein aggregate from carbamoylase overproduction could be alleviated with the aid of GroEL/GroES. In contrast, the production of a high level of d-hydantoinase in an active form can be achieved at low temperature (25 °C) or by the coproduction of DnaJ/DnaK. Overall, with such approaches both recombinant proteins gain more than a four-fold increase in enzyme activity. In addition, by fusion with thioredoxin, d-hydantoinase activity can be increased 25% more than the unfused counterpart in the presence of DnaJ/DnaK. These results indicate the success of our approaches to overproducing d-hydantoinase and carbamoylase in a soluble form in E. coli. Received: 26 November 1999 / Received revision: 28 February 2000 / Accepted: 10 March 2000     

Production of complex viral glycoproteins in plants as vaccine immunogens

Plant molecular farming offers a cost‐effective and scalable approach to the expression of recombinant proteins which has been proposed as an alternative to conventional production platforms for developing countries. In recent years, numerous proofs of concept have established that plants can produce biologically active recombinant proteins and immunologically relevant vaccine antigens that are comparable to those made in conventional expression systems. Driving many of these advances is the remarkable plasticity of the plant proteome which enables extensive engineering of the host cell, as well as the development of improved expression vectors facilitating higher levels of protein production. To date, the only plant‐derived viral glycoprotein to be tested in humans is the influenza haemagglutinin which expresses at ~50 mg/kg. However, many other viral glycoproteins that have potential as vaccine immunogens only accumulate at low levels in planta. A critical consideration for the production of many of these proteins in heterologous expression systems is the complexity of post‐translational modifications, such as control of folding, glycosylation and disulphide bridging, which is required to reproduce the native glycoprotein structure. In this review, we will address potential shortcomings of plant expression systems and discuss strategies to optimally exploit the technology for the production of immunologically relevant and structurally authentic glycoproteins for use as vaccine immunogens.     

Efficient production of secreted proteins by Aspergillus : progress, limitations and prospects

Filamentous fungi are widely used for the production of homologous and heterologous proteins but, compared to homologous proteins, the levels of production of heterologous proteins are usually low. During the last 5 years, the levels of production of heterologous proteins have been drastically improved by fusing the corresponding gene to the 3' end of a homologous gene, encoding a well-secreted protein such as glucoamylase. Nevertheless, little research has been carried out to determine the limitations that hamper heterologous protein production. Recently we have carried out a detailed analysis of the levels of production of several proteins and glucoamylase fusion proteins in defined recombinant Aspergillus awamori strains. In this review we will focus on the use of filamentous fungi for the production of heterologous, especially non-fungal, proteins. In particular, the effect of gene-fusion strategies will be reviewed. Furthermore, the remaining limitations in heterologous protein production and suggestions for improvement strategies for overproduction of these protein will be discussed. Received: 5 July 1996 / Accepted: 6 September 1996     

The use of defective Bombyx mori nucleopolyhedrovirus genomes maintained in Escherichia coli for the rapid generation of occlusion-positive and occlusion-negative expression vectors

A system is described for the rapid generation of Bombyx mori nucleopolyhedrovirus (BmNPV)-based expression vectors. A series of novel BmNPV genomes, that include a mini-F replicon and therefore can be maintained in Escherichia coli, have been generated. These genomes lack a portion of the essential ORF1629 gene and cannot replicate independently in insect cells. However, they can be used as parental genomes for the generation of expression vectors by cotransfection with a transfer plasmid that includes an intact ORF1629. Only recombinant viruses that have acquired the ORF1629 gene from the transfer vector, and have therefore also acquired the foreign gene of interest, can replicate after cotransfection. Parental genomes with and without a polyhedrin gene are described, enabling the generation of occlusion-positive and occlusion-negative recombinant viruses. Occlusion-positive expression vectors enable the oral infection of B. mori larvae and can therefore be used for the mass production of a foreign protein in infected insects.     

Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins

One of the most important branches of genetic engineering is the expression of recombinant proteins using biological expression systems. Nowadays, different expression systems are used for the production of recombinant proteins including bacteria, yeasts, molds, mammals, plants, and insects. Yeast expression systems such as Saccharomyces cerevisiae (S. cerevisiae) and Pichia pastoris (P. pastoris) are more popular. P. pastoris expression system is one of the most popular and standard tools for the production of recombinant protein in molecular biology. Overall, the benefits of protein production by P. pastoris system include appropriate folding (in the endoplasmic reticulum) and secretion (by Kex2 as signal peptidase) of recombinant proteins to the external environment of the cell. Moreover, in the P. pastoris expression system due to its limited production of endogenous secretory proteins, the purification of recombinant protein is easy. It is also considered a unique host for the expression of subunit vaccines which could significantly affect the growing market of medical biotechnology. Although P. pastoris expression systems are impressive and easy to use with well-defined process protocols, some degree of process optimization is required to achieve maximum production of the target proteins. Methanol and sorbitol concentration, Mut forms, temperature and incubation time have to be adjusted to obtain optimal conditions, which might vary among different strains and externally expressed protein. Eventually, optimal conditions for the production of a recombinant protein in P. pastoris expression system differ according to the target protein.     

Regulation of gene expression in adeno-associated virus vectors in the brain

Regulated adeno-associated virus (AAV) vectors have broad utility in both experimental and applied gene therapy, and to date, several regulation systems have exhibited a capability to control gene expression from viral vectors over two orders of magnitude. The tetracycline responsive system has been the most used in AAV, although other regulation systems such as RU486- and rapamycin-responsive systems are reasonable options. AAV vectors influence how regulation systems function by several mechanisms, leading to increased background gene expression and restricted induction. Methods to reduce background expression continue to be explored and systems not yet tried in AAV may prove quite functional. Although regulated promoters are often assumed to exhibit ubiquitous expression, the tropism of different neuronal subtypes can be altered dramatically by changing promoters in recombinant AAV vectors. Differences in promoter-directed tropism have significant consequences for proper expression of gene products as well as the utility of dual vector regulation. Thus regulated vector systems must be carefully optimized for each application.     

Heterologous protein production in methylotrophic yeasts

The facultative methylotrophic yeasts Candida boidinii, Pichia methanolica, Pichia pastoris and Hansenula polymorpha have been developed as systems for heterologous gene expression. They are based on strong and regulatable promoters for expression control derived from methanol metabolism pathway genes. An increasing number of biotechnological applications attest to their status as preferred options among the various gene expression hosts. The well-established P. pastoris and H. polymorpha systems have been utilized in especially competitive and consistent industrial-scale production processes. Pharmaceuticals and technical enzymes produced in these methylotrophs have either already entered the market or are expected to do so in the near future. The article describes the present status of the methylotrophic yeasts as expression systems, focusing on applied examples of the recent past. Received: 9 May 2000 / Received revision: 20 June 2000 / Accepted: 23 June 2000     

<Emphasis Type="Italic">Bacillus megaterium</Emphasis>—from simple soil bacterium to industrial protein production host

Bacillus megaterium has been industrially employed for more than 50 years, as it possesses some very useful and unusual enzymes and a high capacity for the production of exoenzymes. It is also a desirable cloning host for the production of intact proteins, as it does not possess external alkaline proteases and can stably maintain a variety of plasmid vectors. Genetic tools for this species include transducing phages and several hundred mutants covering the processes of biosynthesis, catabolism, division, sporulation, germination, antibiotic resistance, and recombination. The seven plasmids of B. megaterium strain QM B1551 contain several unusual metabolic genes that may be useful in bioremediation. Recently, several recombinant shuttle vectors carrying different strong inducible promoters and various combinations of affinity tags for simple protein purification have been constructed. Leader sequences-mediated export of affinity-tagged proteins into the growth medium was made possible. These plasmids are commercially available. For a broader application of B. megaterium in industry, sporulation and protease-deficient as well as UV-sensitive mutants were constructed. The genome sequence of two different strains, plasmidless DSM319 and QM B1551 carrying seven natural plasmids, is now available. These sequences allow for a systems biotechnology optimization of the production host B. megaterium. Altogether, a “toolbox” of hundreds of genetically characterized strains, genetic methods, vectors, hosts, and genomic sequences make B. megaterium an ideal organism for industrial, environmental, and experimental applications.     

Dramatic Increase in Expression of a Transgene by Insertion of Promoters Downstream of the Cargo Gene

For expression of genes in mammalian cells, various vectors have been developed using promoters including CMV, EF-1α, and CAG promoters and have been widely used. However, such expression vectors sometimes fail to attain sufficient expression levels depending on the nature of cargo genes and/or on host cell types. In the present study, we aimed to develop a potent promoter system that enables high expression levels of cargo genes ubiquitously in many different cell types. We found that insertion of an additional promoter downstream of a cargo gene greatly enhanced the expression levels. Among the constructs we tested, C-TSC cassette (C: CMV-RU5′ located upstream; TSC: another promoter unit composed of triple tandem promoters, hTERT, SV40, and CMV, located downstream of the cDNA plus a polyadenylation signal) had the most potent capability, showing far higher efficiency than that of potent conventional vector systems. The results indicate that the new expression system is useful for production of recombinant proteins in mammalian cells and for application as a gene therapeutic measure.