Semliki Forest Virus Expression System Research Articles

ELISA for Molluscum Contagiosum Virus.

Molluscum contagiosum virus (MCV) is a common skin pathogen of children and young adults. Infection with MCV causes benign skin tumors in children and young adults and is mostly self-limiting. In contrast to orthopoxviruses, MCV infections tend to take a subacute clinical course but may persist for up to 12 months. Current numbers for MCV seroprevalence in different geographical areas are based on a variety of historical serological methods from complement fixation assays to MCV ELISAs based on purified MCV virions and MC133 antigen expressed in a Semliki Forest Virus expression system. A standardized ELISA for the assessment of MCV seroprevalence would be useful to determine global MCV seroprevalence. The methods described show that polypeptides derived from MCV open reading frames MC084 (residues V123 to R230 and V33 to G117), mc133 (residues M1 to N370), and glutathione S-transferase (GST)-H3L (residues I142 to W251) expressed in E.coli RIL+ as GST fusion proteins can be used to assess antibody binding in a GST capture ELISA. We show how the ELISA can be used to screen a panel of patient sera previously characterized with the mc084 V123-R230 ELISA. © 2017 by John Wiley & Sons, Inc.

A Semliki Forest virus expression system as a model for investigating the nuclear import and export of hepatitis B virus nucleocapsid protein.

HBV core protein (HBc), the major component of nucleocapsid is known to have an essential role in the virus life cycle as the transporter of virus genome to the host nucleus; however, molecular details of the intracellular transport of HBc remain unknown. In this study, we investigated the intracellular distribution of the HBc protein resulting from Semliki Forest virus (SFV)-driven HBc gene and HBV RNA pregenome (pgRNA) expression in BHK-21 cells. Fluorescent confocal microscopy revealed extensive HBc protein synthesis in the cytoplasm 12 hr post infection (p.i.) with recombinant pSFV1/HBc or pSFV1/HBVpgRNA viruses. Twenty-four hr p.i., the HBc protein showed asymmetric, predominantly nuclear localization in most cells. However, 42 hr p.i., the number of cells containing intranuclear HBc protein, dramatically decreased to ~ 5%, while cytoplasmic HBc protein was detected in all cells. Remarkably, 24 hr p.i. with pSFV1/HBVpgRNA virus, cytoplasmic HBc protein colocalized with HBs protein. We conclude that a HBs-HBc interaction partially prevents the transport of HBc from the cytoplasm to nucleus. The SFV-driven system can be used for identification of new factors involved in the HBV nuclear import and export.

A simple and efficient method for the production of human glycosylated glial cell line-derived neurotrophic factor using a Semliki Forest virus expression system

Human glial cell line-derived neurotrophic factor (hGDNF) is a very promising protein for the treatment of Parkinson's disease and other neurodegenerative disorders. The present work describes a quick and simple method to obtain a high amount of purified hGDNF using a mammalian cell-derived system. The method is based on the high expression level provided by a Semliki Forest virus vector and its ability to induce a strong shut-off of host-cell protein synthesis in mammalian cells. As a result, hGDNF is the only protein present in the supernatant and can be efficiently purified by a single chromatographic step. Using this system it was possible to eliminate other secreted proteins from the culture medium, like insulin-like growth factor-5, which are hard to remove using other hGDNF production methods. Purified hGDNF presents a complex glycosylation pattern typical of mammalian expression systems and is biologically active. This protocol could be extended to other secreted proteins and could be easily scaled up for industrial purposes.

Heterologously Expressed GLT-1 Associates in ∼200-nm Protein-Lipid Islands

The glutamate transporter GLT-1 from Rattus norvegicus was expressed at high level in baby hamster kidney (BHK-21) cells by the Semliki Forest Virus expression system. We examined the expressed GLT-1 in the plasma membrane and found that the transporter accumulates in detergent-insoluble lipid-protein assemblies. Freeze-fracture, immunogold labeling, and electron microscopy revealed that GLT-1 forms ∼200-nm protein-rich islands in the plasma membrane. Cholesterol depletion in living cells resulted in a dispersion of the GLT-1 islands, indicating that they are the result of lipid-protein rather than protein-protein interactions. Disruption of GLT-1 islands and dispersion of GLT-1 goes along with a reduction of the glutamate transport activity. Our direct visualization of lipid-protein islands in the plasma membrane of tissue culture cells suggests that the reported clustering of glutamate transporters and their cholesterol-dependent transport activity in cells is likewise connected to their association with cholesterol-rich microdomains in the plasma membrane.

Amino-Terminal Processing of the Human Cannabinoid Receptor 1

The human cannabinoid receptor 1 (CB1), a G protein-coupled receptor (GPCR), translocates its long amino-terminal (N-terminal) domain across the endoplasmic reticulum (ER) membrane in a C-to-N terminal direction. Using the Semliki Forest virus (SFV) expression system, CB1 was expressed in baby hamster kidney (BHK) cells. It was found that a large fraction of the CB1 molecules were N-terminally truncated prior to ER translocation. Truncation was fast and independent of the proteasome. It is concluded that the truncation process might be a way to create a novel type of CB1.

Envelope Protein Requirements for the Assembly of Infectious Virions of Porcine Reproductive and Respiratory Syndrome Virus

Virions of porcine reproductive and respiratory syndrome virus (PRRSV) contain six membrane proteins: the major proteins GP5 and M and the minor proteins GP2a, E, GP3, and GP4. Here, we studied the envelope protein requirements for PRRSV particle formation and infectivity using full-length cDNA clones in which the genes encoding the membrane proteins were disrupted by site-directed mutagenesis. By transfection of RNAs transcribed from these cDNAs into BHK-21 cells and analysis of the culture medium using ultracentrifugation, radioimmunoprecipitation, and real-time reverse transcription-PCR, we observed that the production of viral particles is dependent on both major envelope proteins; no particles were released when either the GP5 or the M protein was absent. In contrast, particle production was not dependent on the minor envelope proteins. Remarkably, in the absence of any one of the latter proteins, the incorporation of all other minor envelope proteins was affected, indicating that these proteins interact with each other and are assembled into virions as a multimeric complex. Independent evidence for such complexes was obtained by coexpression of the minor envelope proteins in BHK-21 cells using a Semliki Forest virus expression system. By analyzing the maturation of their N-linked oligosaccharides, we found that the glycoproteins were each retained in the endoplasmic reticulum unless expressed together, in which case they were collectively transported through the Golgi complex to the plasma membrane and were even detected in the extracellular medium. As the PRRSV particles lacking the minor envelope proteins are not infectious, we hypothesize that the virion surface structures formed by these proteins function in viral entry by mediating receptor binding and/or virus-cell fusion.

High-yield Expression, Reconstitution and Structure of the Recombinant, Fully Functional Glutamate Transporter GLT-1 from Rattus norvegicus

The glutamate transporter GLT-1 from Rattus norvegicus was expressed at high level in BHK cells using the Semliki Forest virus expression system. BHK cells infected with viral particles carrying the GLT-1 gene exhibited 30-fold increased aspartate uptake compared to control cells. The expression level of GLT-1 as determined by binding of labelled substrate to membrane preparations was about 3.5 x 10(6) functional transporters per cell, or 61 pmol GLT-1 per milligram of membrane protein. Purification of the His-tagged protein by Ni2+-NTA affinity chromatography enabled the routine production and purification of milligram quantities of fully functional transporter. Transport activity required reducing conditions and the addition of extra lipid throughout the purification. The apparent molecular mass of the recombinant transporter was 73 kDa or 55 kDa, corresponding to the glycosylated and non-glycosylated form, respectively. Both forms were active upon separation on a lectin column and reconstitution into liposomes. Glycosylated and non-glycosylated GLT-1 were transported to the plasma membrane with equal efficiency. Our results show that N-glycosylation does not affect the trafficking or the transport activity of GLT-1. The low-resolution structure of GLT-1 was determined by electron microscopy and single particle reconstruction.

Annexin 2 Promotes the Formation of Lipid Microdomains Required for Calcium-regulated Exocytosis of Dense-Core Vesicles

Annexin 2 is a calcium-dependent phospholipid-binding protein that has been implicated in a number of membrane-related events, including regulated exocytosis. In chromaffin cells, we previously reported that catecholamine secretion requires the translocation and formation of the annexin 2 tetramer near the exocytotic sites. Here, to obtain direct evidence for a role of annexin 2 in exocytosis, we modified its expression level in chromaffin cells by using the Semliki Forest virus expression system. Using a real-time assay for individual cells, we found that the reduction of cytosolic annexin 2, and the consequent decrease of annexin 2 tetramer at the cell periphery, strongly inhibited exocytosis, most likely at an early stage before membrane fusion. Secretion also was severely impaired in cells expressing a chimera that sequestered annexin 2 into cytosolic aggregates. Moreover, we demonstrate that secretagogue-evoked stimulation triggers the formation of lipid rafts in the plasma membrane, essential for exocytosis, and which can be attributed to the annexin 2 tetramer. We propose that annexin 2 acts as a calcium-dependent promoter of lipid microdomains required for structural and spatial organization of the exocytotic machinery.

Translation of hepatitis B virus (HBV) surface proteins from the HBV pregenome and precore RNAs in Semliki Forest virus-driven expression.

Hepatitis B virus (HBV) pregenome RNA (pgRNA) serves as a translation template for the HBV core (HBc) protein and viral polymerase (Pol). HBV precore RNA (pcRNA) directs the synthesis of the precore (preC) protein, a precursor of the hepatitis B e antigen (HBeAg). pgRNA and pcRNA were expressed in the Semliki Forest virus (SFV) expression system. Besides the HBc and preC proteins, there was revealed the synthesis of all three forms of HBV surface (HBs) proteins: long (LHBs), middle (MHBs) and short (SHBs), the start codons of which are located more than 1000 nt downstream of the HBc and preC start codons. Moreover, other HBV templates, such as 3'-truncated pgRNA lacking 3' direct repeat and Pol mRNA, both carrying internally the HBs sequences, provided the synthesis of three HBs protein forms in the SFV-driven expression system. Maximal production of the HBs was provided by Pol mRNA, while HBc- and preC-producing templates showed relatively low internal translation of the HBs. These data allow the proposal of a ribosome leaky scanning model of internal translation initiation for HBs proteins. The putative functional role of such exceptional synthesis of the HBs proteins from the pgRNA and pcRNA templates in the natural HBV infection process needs further evaluation.

Effect of intranasal administration of semliki forest virus recombinant particles expressing reporter and cytokine genes on the progression of experimental autoimmune encephalomyelitis

We have initiated studies to determine the feasibility of employing the Semliki Forest virus (SFV) expression system as a central nervous system (CNS) vector. We investigated the effects of infecting Balb/c mice intranasally (i.n.) with recombinant SFV particles expressing the enhanced green fluorescent protein (EGFP) reporter gene. EGFP expression was detected by fluorescence microscopy in the olfactory bulb as early as 1 day postinfection. No pathological changes were associated with infection. Viral RNA could be detected in the olfactory mucosa only, whereas fluorescence was detected in axons in the olfactory bulb, indicating that only the expressed protein was present. A vector expressing interleukin 10 (IL-10) was constructed and shown to induce good cytokine expression in cultured cells. IL-10 expression in the nasal passage and olfactory bulb of infected mice was enhanced following i.n. administration of such particles. Mice induced for experimental autoimmune encephalomyelitis (EAE) were treated i.n. with vectors expressing EGFP and IL-10 and with empty vector. The EGFP-expressing and empty vectors were found to exacerbate EAE, whereas that expressing IL-10 ameliorated EAE. It is concluded that the mice showed a significant biological response when treated i.n. with recombinant SFV particles and that such particles administered by the i.n. route have potential as a noninvasive vector for protein delivery to the CNS.

Role of actin filaments in targeting of Crimean Congo hemorrhagic fever virus nucleocapsid protein to perinuclear regions of mammalian cells.

Crimean-Congo hemorrhagic fever virus is the causative agent of a severe disease throughout Africa, Europe, and Asia. Like other members of the genus Nairovirus, the Crimean-Congo hemorrhagic fever virus contains three genomic RNA segments, the small (S), medium (M), and large (L) segments. The S segment encodes the viral nucleocapsid protein (NP), while the M and L segments encode the glycoproteins and the RNA-dependent RNA polymerase, respectively. In this study, the site of expression and assembly of Crimean-Congo hemorrhagic fever virus NP in mammalian cells have been investigated. It was found that the NP is localized in the perinuclear region of infected cells. By using the Semliki forest virus expression system, it was shown that the Crimean-Congo hemorrhagic fever virus NP is targeted to the perinuclear region of cells in the absence of native RNA segments and virally encoded glycoproteins. It was also demonstrated that the Crimean-Congo hemorrhagic fever virus NP was not expressed as a Golgi-membrane associated protein. By using Cytochalasin D, an agent that disrupts actin filaments, it was found that actin filaments are involved in targeting the viral NP to perinuclear regions. We also demonstrated that disruption of actin filaments reduced the assembly of infectious Crimean-Congo hemorrhagic fever virus up to 97%. Furthermore, we showed that the NP of Crimean-Congo hemorrhagic fever virus NP interacts with actin.

Overexpression of the myelin proteolipid protein leads to accumulation of cholesterol and proteolipid protein in endosomes/lysosomes: implications for Pelizaeus-Merzbacher disease.

Duplications and overexpression of the proteolipid protein (PLP) gene are known to cause the dysmyelinating disorder Pelizaeus-Merzbacher disease (PMD). To understand the cellular response to overexpressed PLP in PMD, we have overexpressed PLP in BHK cells and primary cultures of oligodendrocytes with the Semliki Forest virus expression system. Overexpressed PLP was routed to late endosomes/lysosomes and caused a sequestration of cholesterol in these compartments. Similar results were seen in transgenic mice overexpressing PLP. With time, the endosomal/lysosomal accumulation of cholesterol and PLP led to an increase in the amount of detergent-insoluble cellular cholesterol and PLP. In addition, two fluorescent sphingolipids, BODIPY–lactosylceramide and –galactosylceramide, which under normal conditions are sorted to the Golgi apparatus, were missorted to perinuclear structures. This was also the case for the lipid raft marker glucosylphosphatidylinositol–yellow fluorescence protein, which under normal steady-state conditions is localized on the plasma membrane and to the Golgi complex. Taken together, we show that overexpression of PLP leads to the formation of endosomal/lysosomal accumulations of cholesterol and PLP, accompanied by the mistrafficking of raft components. We propose that these accumulations perturb the process of myelination and impair the viability of oligodendrocytes.

Recombinant Semliki Forest virus vector exhibits potential for avian virus vaccine development

The Semliki Forest virus (SFV) expression system was evaluated as a basis for avian vaccine development. Initial studies indicated that 1-day-old specific pathogen-free (SPF) chicks were susceptible to infection with an infectious strain of SFV, producing SFV-specific antibodies but no clinical disease. One-day-old SPF chicks immunised intramuscularly with recombinant replication-defective SFV (rSFV) particles expressing the Escherichia coli (E. coli) lacZ reporter gene developed high titres of β-gal- specific antibodies at 4 weeks p.i. after two inoculations. In contrast, significantly lower antibody levels were elicited in chicks immunised with a recombinant SFV-based DNA construct or a conventional CMV promoter-based DNA plasmid. rSFV particles encoding the protective VP2 protein or the VP2/VP4/VP3 polyprotein of infectious bursal disease virus (IBDV) were produced and the expressed antigens were characterised in cell culture. Proteins of the correct size were generated and found to react against a range of IBDV-specific monoclonal antibodies. Immunisation of 1-day-old SPF chicks with rSFV particles encoding the IBDV proteins resulted in specific antibodies being elicited in all birds, neutralising antibodies being induced in some but not all birds.

Tomato Spotted Wilt Virus Glycoproteins Exhibit Trafficking and Localization Signals That Are Functional in Mammalian Cells

The glycoprotein precursor (G1/G2) gene of tomato spotted wilt virus (TSWV) was expressed in BHK cells using the Semliki Forest virus expression system. The results reveal that in this cell system, the precursor is efficiently cleaved and the resulting G1 and G2 glycoproteins are transported from the endoplasmic reticulum (ER) to the Golgi complex, where they are retained, a process that could be blocked by tunicamycin. Expression of G2 alone resulted in transport to and retention in the Golgi complex, albeit less efficient, suggesting that G2 contains a Golgi retention signal. G1 alone was retained in the ER, irrespective of whether it contained the precursor's signal sequence or its own N-terminal hydrophobic sequence. Coexpression of G1 and G2 from separate gene constructs resulted in rescue of efficient G1 transport, as the proteins coaccumulated in the Golgi complex, indicating that their interaction is essential for proper targeting to this organelle. The results demonstrate that transport and targeting of the plant TSWV glycoproteins in mammalian BHK cells are strikingly similar to those of animal-infecting bunyavirus glycoproteins in mammalian cells. The observations are likely to reflect the dual tropism of TSWV, which replicates both in its plant host and in its animal (thrips) vector.

Intracellular localization of vaccinia virus extracellular enveloped virus envelope proteins individually expressed using a Semliki Forest virus replicon.

The extracellular enveloped virus (EEV) form of vaccinia virus is bound by an envelope which is acquired by wrapping of intracellular virus particles with cytoplasmic vesicles containing trans-Golgi network markers. Six virus-encoded proteins have been reported as components of the EEV envelope. Of these, four proteins (A33R, A34R, A56R, and B5R) are glycoproteins, one (A36R) is a nonglycosylated transmembrane protein, and one (F13L) is a palmitylated peripheral membrane protein. During infection, these proteins localize to the Golgi complex, where they are incorporated into infectious virus that is then transported and released into the extracellular medium. We have investigated the fates of these proteins after expressing them individually in the absence of vaccinia infection, using a Semliki Forest virus expression system. Significant amounts of proteins A33R and A56R efficiently reached the cell surface, suggesting that they do not contain retention signals for intracellular compartments. In contrast, proteins A34R and F13L were retained intracellularly but showed distributions different from that of the normal infection. Protein A36R was partially retained intracellularly, decorating both the Golgi complex and structures associated with actin fibers. A36R was also transported to the plasma membrane, where it accumulated at the tips of cell projections. Protein B5R was efficiently targeted to the Golgi region. A green fluorescent protein fusion with the last 42 C-terminal amino acids of B5R was sufficient to target the chimeric protein to the Golgi region. However, B5R-deficient vaccinia virus showed a normal localization pattern for other EEV envelope proteins. These results point to the transmembrane or cytosolic domain of B5R protein as one, but not the only, determinant of the retention of EEV proteins in the wrapping compartment.

Production of human UDP-glucuronosyltransferases 1A6 and 1A9 using the Semliki Forest virus expression system

Human UDP-glucuronosyltransferases (UGTs) 1A6 and 1A9 were expressed using Semliki Forest virus (SFV) vectors. Infection of chinese hamster lung fibroblast V79 cells with recombinant SFV-UGT viruses resulted in efficient protein expression as detected by metabolic labeling, Western blot analyses and immunofluorescence microscopy. The expression of UGT1A6 and UGT1A9 in the SFV-infected cells was approximately two fold higher than in a stable V79 cell line. No UGT signal was detected in noninfected cells. In addition, SFV-UGT viruses also efficiently infected other mammalian cells, such as baby hamster kidney (BHK), chinese hamster ovary (CHO) and human lung (WI-26 VA4) cells leading to high production of recombinant enzyme. The measurement of enzyme activities and kinetic parameters using p-nitrophenol and nitrocatechol (entacapone) as substrates for UGT1A6 and UGT1A9, respectively, showed that the overall kinetic properties of the enzymes produced by the two systems were similar. We conclude that the SFV expression system represents an efficient, fast and versatile method for production of metabolic enzymes for in vitro assays.

ATP is required for correct folding and disulfide bond formation of rotavirus VP7.

Rotavirus is one of very few viruses that utilize the endoplasmic reticulum (ER) for assembly, and therefore it has been used as an attractive model to study ER-associated protein folding. In this study, we have examined the requirements for metabolic energy (ATP) for correct folding of the luminal and ER-associated VP7 of rotavirus. We found that VP7 rapidly misfolds in an energy-depleted milieu and is not degraded within 60 min. We also found that VP7 attained a stable minimum-energy state soon after translation in the ER. Most surprisingly, energy-misfolded VP7 could be recovered and establish correct disulfide bonds and antigenicity following a shift to an ATP-rich milieu. Using a Semliki Forest virus expression system, we observed that VP7 requires ATP and cellular, but not viral, factors for correct disulfide bond formation. Our results show for the first time that the disulfide bond formation of rotavirus VP7 is an ATP-dependent process. It has previously been shown that chaperones hydrolyze ATP during interaction with newly synthesized polypeptides and prevent nonproductive intra- and intermolecular interactions. The most reasonable explanation for the energy requirement of VP7 is thus a close interaction during folding with an ATP-dependent chaperone, such as BiP (Grp78), and possibly with protein disulfide isomerase. Taken together, our observations provide new information about folding of ER-associated proteins in general and rotavirus VP7 in particular.

Selective targeting of avidin/mannose 6-phosphate receptor chimeras to early or late endosomes

In this study we have used the Semliki forest virus expression system to transiently express chimeric proteins that contain transmembrane and cytoplasmic domains of the cation-independent mannose 6-phosphate receptor (CI-MPR) fused to chicken avidin. Immunofluorescence and electron microscopy studies showed that the chimeric protein with the entire cytoplasmic domain of CI-MPR was transported to late endosomes, where it accumulated. We made use of the biotin-binding capacity of lumenal avidin, and found that, in agreement with this distribution, the chimeric protein could be labelled with biotinylated HRP endocytosed for a long, but not a brief, period of time. However, truncation of the C-terminal tail distal to the rapid endocytosis motif (YKYSKV), caused the truncated chimera to be transported to, and accumulated within, early endosomes. This truncated chimera did not reach recycling early endosomes labelled with internalised transferrin, to any significant extent, but was accessible to biotinylated HRP internalised for 5 min (or for longer periods at 19 degrees C). Coinfection of these chimeras showed that they follow the same route from the TGN to the early endosomes. We conclude that the sequence distal to the endocytosis motif contains the signals which are required for efficient transport to late endosomes. Our results also suggest that the YKYSKV sequence close to the CI-MPR transmembrane segment is sufficient for targeting to sorting early endosomes.

Cell Culture Evaluation of the Semliki Forest Virus Expression System As a Novel Approach for Antigen Delivery and Expression in Fish.

Heterologous gene expression by Semliki Forest virus (SFV) expression vectors was investigated in fish cell culture. Experiments performed using an infectious strain of SFV, replication-defective SFV particles, and recombinant SFV RNA constructs encoding the Escherchia coli LacZ or firefly luciferase reporter genes indicated that levels of SFV-mediated expression in fish cells were dependent on cell type and temperature. Maximal expression levels were observed in the two salmonid-derived cell lines CHSE-214 and F95/9 at 25 degrees C and 20 degrees C. As the temperature was lowered to 15 degrees C or below, levels of reporter gene expression were reduced up to 1000-fold, indicating that the SFV replication complex functioned inefficiently at low temperatures. The ability of SFV expression systems to function in fish cells was further investigated by analyzing the expression of the protective VP2 antigen of infectious pancreatic necrosis virus (IPNV) from the various constructs, including a novel DNA-based SFV plasmid. The VP2 protein produced in CHSE-214 and F95/9 cells transfected or infected with the recombinant SFV-IPNV VP2 constructs appeared to be synthesized in an antigenically correct form, as evidenced by the ability to react with several conformation-dependent IPNV-specific monoclonal antibodies. Whether the temperature-restricted replication and expression displayed by SFV-based constructs in fish cell culture also occurs in vivo remains to be determined.

Large scale transient 5-HT3 receptor production with the Semliki Forest Virus Expression System.

The expression of recombinant proteins with the Semliki Forest Virus (SFV) system has been scaled up to bioreactor scale. As a model protein for this study the human 5-HT(3) receptor was chosen. The gene for the receptor was subcloned into the SFV expression plasmid pSFV1. Virus production by in vivo packaging and production of the recombinant protein was scaled up, the latter to a reactor volume of 11.5 l. A Vibromix(TM) agitation system was chosen to overcome aggregation problems of BHK cells in suspension. In the process, cells were first grown to a density of 10(6) cells/ml, the medium was then exchanged with fresh medium and the culture was infected with the recombinant virus at an estimated multiplicity of infection of 30. 24 h post infection we measured an expression level of 3 million functional 5-HT(3) receptors per cell. For harvesting, the cells were pelleted by centrifugation. The receptor protein was purified in a single step (Hovius et al., 1998) by exploiting the hexa-His tag at minimal protein loss (51% yield). Experiments to optimise expression resulted in yields up to 8 million receptors per cell, when the pH of a suspension culture was controlled at pH 7.3. Rapid virus generation and protein production, high protein yields as well as successful large scale application have made the SFV expression system attractive to produce large quantities of recombinant protein in a very short time. After optimisation of the expression conditions (in particular by setting the pH at 7.3), yields were increased twofold.