Cloning of the GSH1 and GSH2 Genes Complementing the Defective Biosynthesis of Glutathione in the Methylotrophic Yeast Hansenula polymorpha

The cloning of 7.2- and 9.6-kbp fragments of the methylotrophic yeast Hansenula polymorpha DNA restored the wild-type phenotype Gsh⁺ in the glutathione-dependent gsh1 and gsh2 mutants of this yeast defective in glutathione (GSH) synthesis because of a failure of the -glutamylcysteine synthetase reaction. The 9.6-kbp DNA fragment was found to contain a 4.3-kbp subfragment, which complemented the Gsh^– phenotype of the gsh2 mutant. The Gsh⁺ transformants of the gsh1 and gsh2 mutants, which bear plasmids pG1 and pG24, having the 7.2- and 4.3-kbp DNA fragments, respectively, had a completely restored wild-type phenotype with the ability to synthesize GSH and to grow in GSH-deficient synthetic media on various carbon sources, including methanol, and with acquired tolerance to cadmium ions. In addition, the 4.3-kbp DNA fragment borne by plasmid pG24 eliminated pleiotropic changes in the gsh2 mutants associated with methylotrophic growth in a semisynthetic (GSH-supplemented) medium (poor growth and alterations in the activity of the GSH-catabolizing enzyme -glutamyltransferase and the methanol-oxidizing enzyme alcohol oxidase).     

Production of recombinant hirudin in Hansenula polymorpha: variation of gene expression level depends on methanol oxidase and fermentation strategies

Various recombinant Hansenula polymorpha strains were developed and compared for their level of expression of the anticoagulant hirudin. H. polymorpha DL1-57 harboring an autonomously replicating sequence, HARS36, efficiently expressed the gene for recombinant hirudin. The effect of methanol oxidase (MOX) on the expression of the hirudin gene in H. polymorpha DL1-57 was studied, and the fermentation strategies coupled with the MOX activity and an antioxidant, tocopherol, were also examined. Received 4 February 1998/ Accepted in revised form 24 June 1998     

Methanol utilization by a strain of Aspergillus niger: influence on the synthesis and activity of pectinases

During the production of pectinases by a strain of Aspergillus niger isolated from rotten lemons, methanol was liberated into the medium due to the cleavage of the pectin molecule used as the carbon source. The methanol was subsequently consumed by the microorganism but neither the synthesis nor the activity of pectinesterase and polygalacturonase was affected. Although not studied in detail, the mechanism involved in the utilization of methanol is similar to that described for methylotrophic yeasts.     

Effect of the levels of dissolved oxygen on the expression of recombinant proteins in four recombinantEscherichia coli strains

Summary Four recombinant strains ofEscherichia coli were examined for the effects of the dissolved oxygen level on the level of biomass, the plasmid content, and the level of recombinant protein at the stationary phase of batch growth. Strains JM101/pYEJ001, and TB-1/pYEJ001 (encoding chloramphenicol acetyltransferase), and strain TB-1/p1034, and TB-1/pUC19 (encoding -galactosidase) were grown at the constant dissolved oxygen levels of 0, 50, and 100% air saturation, as well as in the absence of dissolved, oxygen control. The biomass of all strains under constant aerobic conditions was 12–36 times higher than that under anaerobic conditions, but was the same as or slightly higher than that without dissolved oxygen control. The plasmid content in all strains under anaerobic conditions was 2.9–11.7 times higher than that under aerobic conditions. The optimal dissolved oxygen concentration for the specific activity of recombinant proteins was dependent upon the strain. In no strain were constant aerobic conditions optimal. However, because of the effect on biomass, controlled aerobic conditions were optimal for the volumetric activity of recombinant protein in all but one strain.     

Use of GFP fusions for the isolation of Escherichia coli strains for improved production of different target recombinant proteins

High level expression of a recombinant gene results in growth arrest, followed by overgrowth by non-productive derivatives. Two methods are described for the isolation of E. coli BL21* strains that are improved hosts for recombinant protein production. Both are based upon the observations (i) that fluorescence of a C-terminal GFP tag is a reliable reporter of the production and correct folding of the N-terminal target domain; and (ii) rare mutants arise spontaneously that remain productive during long periods of high level recombinant protein production. The first method relies upon identifying these mutants amongst colonies on agar plates; the other exploits fluorescence activated cell sorting. Although identical mutations in the regulatory region of the T7 polymerase gene were found in all of the improved host strains isolated, they differed in their ability to accumulate the outer membrane protein, Ccp, or a cytoplasmic protein, CheY-GFP. Cytochrome c peroxidase activity of recombinant Ccp from one of these strains was demonstrated. Changes in levels of T7 polymerase expression are therefore insufficient to ensure increased accumulation of all recombinant proteins. We demonstrate that the methods described allow strains to be isolated that carry other, currently uncharacterised mutations that are required depending on the target protein.     

Factors affecting the expression of foreign proteins inEscherichia coli

Summary A variety of factors affect the expression of foreign proteins inEscherichia coli. These include: promoter strength, efficiency of ribosome binding, stability of the foreign protein inE. coli, location of the foreign protein inE. coli, the codons used to encode the foreign protein, the metabolic state of the cell, and the location, stability and copy number of the foreign gene. This paper contains a critical review of these factors with the idea that a detailed understanding of them is the key to the development of strategies for the efficient large-scale production of foreign proteins inE. coli.     

Effect of salinity on the adaptive capacity of recombinant strains ofEscherichia coli andBacillus subtilis

The effect of different concentrations of salts on natural and recombinant strains ofBacillus subtilis andEscherichia coli was studied. The recombinant strain ofB. subtilis was found to be more osmotolerant than the wild-type strain of this bacterium, whereas the opposite situation was observed for the recombinant and wild-type strains ofE. coli. Some salts exerted a bacteriostatic effect onE. coli andB. subtilis. The adaptive capacity of recombinant strains depended on the number of plasmid copies in the cells. The introduction of recombinant bacteria into model ecosystems resulted in the generation of their variants with increased osmotolerance.     

Some factors influencing the proportion of periplasmic hepatitisB virus pre-S2 antigen in the recombinant yeastHansenula polymorpha

Summary A central composite design (CCD) was used to evaluate, for the purpose of future process optimization, the influence of pH, yeast extract and ammonium chloride concentrations on the proportion of periplasmic hepatitisB pre-S2 antigen in the recombinant yeastHansenula polymorpha. Each factor was tested at five levels, and a second order polynomial model for the proportion of periplasmic antigen was fitted to the treatment combinations. pH showed the greatest effect: the proportion of periplasmic antigen was greatly increased at the higher pH levels. At the higher pH levels used, the proportion of periplasmic antigen was enhanced by a high concentration of ammonium chloride. Additional experiments have confirmed both the validity of the selected model and the optimal conditions found. A significant correlation was found between the proportion of periplasmic antigen and the total yield of antigen. These results indicated that is should be possible to modulate the distribution of the pre-S2 antigen between the periplasm and the cytoplasm of the yeast.     

Challenges and opportunities in the purification of recombinant tagged proteins

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs “tag-ligand” combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established “tag-ligand” systems available for fusion protein purification and also explores current unconventional strategies under development.     

Elastin-like polypeptide fusions enhance the accumulation of recombinant proteins in tobacco leaves

The production of recombinant proteins in plants is an active area of research and many different high-value proteins have now been produced in plants. Tobacco leaves have many advantages for recombinant protein production particularly since they allow field production without seeds, flowers or pollen and therefore provide for contained production. Despite these biosafety advantages recombinant protein accumulation in leaves still needs to be improved. Elastin-like polypeptides are repeats of the amino acids “VPGXG” that undergo a temperature dependant phase transition and have utility in the purification of recombinant proteins but can also enhance the accumulation of recombinant proteins they are fused to. We have used a 11.3 kDa elastin-like polypeptide as a fusion partner for three different target proteins, human interleukin-10, murine interleukin-4 and the native major ampullate spidroin protein 2 gene from the spider Nephila clavipes. In both transient analyses and stable transformants the concentrations of the fusion proteins were at least an order of magnitude higher for all of the fusion proteins when compared to the target protein alone. Therefore, fusions with a small ELP tag can be used to significantly enhance the accumulation of a range of different recombinant proteins in plant leaves. An erratum to this article can be found at     

A highly efficient integrated chromatographic procedure for the purification of recombinant hepatitis B surface antigen from Hansenula polymorpha

The high expression level of recombinant hepatitis B surface antigen obtained from Hansenula polymorpha yeast cell (Hans-HBsAg) made it possible to produce HBsAg vaccine in a large scale and by cost-effective process. However, the present available purification process was somewhat tedious, time-consuming and difficult to scale up. To improve the purification efficiency and simplify the purification process, an integrated chromatographic process was developed and optimized. The downstream process included ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and gel filtration chromatography (GFC). A series of chromatographic adsorbents were evaluated for their performances on the purification of Hans-HBsAg, and then the suitable adsorbents for IEC and HIC were screened out, respectively. After clarification by centrifugation, the supernatant of cell disruption (SCD) was purified by standard chromatographic steps, IEC on DEAE Sepharose FF, HIC on Butyl-S-QZT and GFC on Sepharose 4FF. Furthermore, HBsAg recovery, purification factor (PF) and purity during the downstream process were evaluated with enzyme-linked immunosorption assay (ELISA), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance size-exclusion chromatography (HPSEC). The results demonstrated that in the scale of 550ml SCD, the total HBsAg recovery and PF of the whole procedure were about 21.0+/-0.9% and 80.7+/-8.4 (n=3) respectively, with the purity of above 99%. This new downstream process was efficient, reproducible and relatively easy to be scaled up.     

A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids

A plasmid cloning vector with ampicillin-resistance and streptomycin-sensitivity markers is suitable for the direct selection of strains carrying recombinant plasmids. The selection for plasmid transformants utilizes their ampicillin resistance whereas selection for recombinant plasmids is based on the inactivation of the rpsL gene contained on the plasmid. When streptomycin-resistant Escherichia coli strains are used as recipients in transformation, transformants carrying the parental plasmid are phenotypically sensitive to streptomycin while those carrying hybrid plasmids are resistant to streptomycin.     

Transgenic Brassica carinata as a vehicle for the production of recombinant proteins in seeds

Hirudin, a blood anticoagulant protein from leeches, and β-glucuronidase were produced in Brassica carinata Braun (Ethiopian mustard) seeds using oleosin as a carrier. Cotyledonary petioles were infected with Agrobacterium strains containing oleosin-glucuronidase (pCGNOBPGUS-A) or oleosin-hirudin (pCGN-OBHIRT) constructs. Polymerase chain reaction and neomycin phosphotransferase II enzyme assays confirmed the presence of the fusion genes in plants regenerating under selection. The fusion polypeptides were correctly expressed and targeted to the oil-bodies of the seeds with high fidelity (ca. 90%). Recombinant protein was purified from all other cellular protein by a simple flotation process and cleaved from oil-bodies using the endoprotease, Factor Xa. Hirudin activity was measured using a colorimetric thrombin inhibition assay and an activity in the range of 0.2–0.4 antithrombin units per milligram of oil-body protein was detected. B. carinata offers an attractive alternative for the production of recombinant proteins using oleosin technology. Received: 20 March 1997 / Revision received: 5 June 1997 / Accepted: 30 July 1997     

Fourier transform infrared spectroscopy analysis of the conformational quality of recombinant proteins within inclusion bodies

The solubility of recombinant proteins produced in bacterial cells is considered a key issue in biotechnology as most overexpressed polypeptides undergo aggregation in inclusion bodies, from which they have to be recovered by solubilization and refolding procedures. Physiological and molecular strategies have been implemented to revert or at least to control aggregation but they often meet only partial success and have to be optimized case by case. Recent studies have shown that proteins embedded in inclusion bodies may retain residual structure and biological function and question the former axiom that solubility and activity are necessarily coupled. This allows for a switch in the goals from obtaining soluble products to controlling the conformational quality of aggregated proteins. Central to this approach is the availability of analytical methods to monitor protein structure within inclusion bodies. We describe here the use of Fourier transform infrared spectroscopy for the structural analysis of inclusion bodies both purified from cells and in vivo. Examples are reported concerning the study of kinetics of aggregation and structure of aggregates as a function of expression levels, temperature and co-expression of chaperones.     

重组鼠疫菌V抗原的纯化及其豚鼠免疫保护力初探

采用Ni^2 亲和层析方法,对用工程化大肠杆菌BL21(DE3)表达的重组鼠疫菌v抗原进行纯化,目标蛋白纯度达到90％以上。以氢氧化铝凝胶配制吸附疫苗,经二针次肌内注射免疫实验豚鼠后,对皮下注射400个致死剂量(MLD)强毒鼠疫菌攻击有一定保护效力,存活率为20％。结果表明,重组鼠疫菌V抗原有望作为改进的F1 V亚单位疫苗的主要成分。     

Early detection of antibodies against various structural proteins of the SARS-associated coronavirus in SARS patients

Severe acute respiratory syndrome (SARS), a new disease with symptoms similar to those of atypical pneumonia, raised a global alert in March 2003. Because of its relatively high transmissibility and mortality upon infection, probable SARS patients were quarantined and treated with special and intensive care. Therefore, instant and accurate laboratory confirmation of SARS-associated coronavirus (SARS-CoV) infection has become a worldwide interest. For this need, we purified recombinant proteins including the nucleocapsid (N), envelope (E), membrane (M), and truncated forms of the spike protein (S1–S7) of SARS-CoV inEscherichia coli. The six proteins N, E, M, S2, S5, and S6 were used for Western blotting (WB) to detect various immunoglobulin classes in 90 serum samples from 54 probable SARS patients. The results indicated that N was recognized in most of the sera. In some cases, S6 could be recognized as early as 2 or 3 days after illness onset, while S5 was recognized at a later stage. Furthermore, the result of recombinant-protein-based WB showed a 90% agreement with that of the whole-virus-based immunofluorescence assay. Combining WB with existing RT-PCR, the laboratory confirmation for SARS-CoV infection was greatly enhanced by 24.1%, from 48.1% (RT-PCR alone) to 72.2%. Finally, our results show that IgA antibodies against SARS-CoV can be detected within 1 week after illness onset in a few SARS patients.     

Comparative analysis of the immune responses induced by native versus recombinant versions of the ASP-based vaccine against the bovine intestinal parasite Cooperia oncophora

The protective capacities of a native double-domain activation-associated secreted protein (ndd-ASP)-based vaccine against the cattle intestinal nematode Cooperia oncophora has previously been demonstrated. However, protection analysis upon vaccination with a recombinantly produced antigen has never been performed. Therefore, the aim of the current study was to test the protective potential of a Pichia-produced double-domain ASP (pdd-ASP)-based vaccine against C. oncophora. Additionally, we aimed to compare the cellular and humoral mechanisms underlying the vaccine-induced responses by the native (ndd-ASP) and recombinant vaccines. Immunisation of cattle with the native C. oncophora vaccine conferred significant levels of protection after an experimental challenge infection, whereas the recombinant vaccine did not. Moreover, vaccination with ndd-ASP resulted in a higher proliferation of CD4-T cells both systemically and in the small intestinal mucosa when compared with animals vaccinated with the recombinant antigen. In terms of humoral response, although both native and recombinant vaccines induced similar levels of antibodies, animals vaccinated with the native vaccine were able to raise antibodies with greater specificity towards ndd-ASP in comparison with antibodies raised by vaccination with the recombinant vaccine, suggesting a differential immune recognition towards the ndd-ASP and pdd-ASP. Finally, the observation that animals displaying antibodies with higher percentages of recognition towards ndd-ASP also exhibited the lowest egg counts suggests a potential relationship between antibody specificity and protection.     

Influences of the recombinant artificial cell adhesive proteins on the behavior of human umbilical vein endothelial cells in serum-free culture

To improve the safety of cellular therapy products, it is necessary to establish a serum-free cell culture method that can exclude animal-derived materials in order to avoid contamination with transmissible agents. It would be optimal if the proteins necessary to a serum-free culture could be provided as recombinant proteins. In this study, the influences of recombinant artificial cell adhesive proteins on the behavior of human umbilical vein endothelial cells (HUVECs) in serum-free culture were examined in comparison with the influence of plasma fibronectin (FN). The recombinant proteins used were Pronectin F (PF), Pronectin F PLUS (PFP), Pronectin L (PL), Retronectin (RN), and Attachin (AN). HUVECs adhered more efficiently on PF or PFP than on FN. No cells adhered on PL. Regarding the VEGF or bFGF-induced cell growth, the cells on PF and PFP proliferated at a similar rate to the cells on FN. RN and AN were less effective in supporting cell growth. Since cell adhesion on PF and PFP induced phosphorylation of focal adhesion kinase, they are thought to activate integrin-mediated intracellular signaling. The cells cultured on PF or PFP were able to produce prostaglandin I(2) or tissue-plasminogen activator in response to thrombin. However, thrombin caused detachment of the cells from PF but not from PFP or FN, meaning that the cells were able to adhere more tightly on PFP or FN than on PF. These data indicate that PFP could be applicable as a substitute for plasma FN.