Cabazitaxel versus abiraterone or enzalutamide in poor prognosis metastatic castration-resistant prostate cancer: a multicentre, randomised, open-label, phase II trial.

BACKGROUND: Treatment of poor prognosis metastatic castration-resistant prostate cancer (mCRPC) includes taxane chemotherapy and androgen receptor pathway inhibitors (ARPI). We sought to determine optimal treatment in this setting. PATIENTS AND METHODS: This multicentre, randomised, open-label, phase II trial recruited patients with ARPI-naive mCRPC and poor prognosis features (presence of liver metastases, progression to mCRPC after <12 months of androgen deprivation therapy, or ≥4 of 6 clinical criteria). Patients were randomly assigned 1 : 1 to receive cabazitaxel plus prednisone (group A) or physician's choice of enzalutamide or abiraterone plus prednisone (group B) at standard doses. Patients could cross over at progression. The primary endpoint was clinical benefit rate for first-line treatment (defined as prostate-specific antigen response ≥50%, radiographic response, or stable disease ≥12 weeks). RESULTS: Ninety-five patients were accrued (median follow-up 21.9 months). First-line clinical benefit rate was greater in group A versus group B (80% versus 62%, P = 0.039). Overall survival was not different between groups A and B (median 37.0 versus 15.5 months, hazard ratio (HR) = 0.58, P = 0.073) nor was time to progression (median 5.3 versus 2.8 months, HR = 0.87, P = 0.52). The most common first-line treatment-related grade ≥3 adverse events were neutropenia (cabazitaxel 32% versus ARPI 0%), diarrhoea (9% versus 0%), infection (9% versus 0%), and fatigue (7% versus 5%). Baseline circulating tumour DNA (ctDNA) fraction above the cohort median and on-treatment ctDNA increase were associated with shorter time to progression (HR = 2.38, P < 0.001; HR = 4.03, P < 0.001). Patients with >30% ctDNA fraction at baseline had markedly shorter overall survival than those with undetectable ctDNA (HR = 38.22, P < 0.001). CONCLUSIONS: Cabazitaxel was associated with a higher clinical benefit rate in patients with ARPI-naive poor prognosis mCRPC. ctDNA abundance was prognostic independent of clinical features, and holds promise as a stratification biomarker.

Navigating systemic therapy for metastatic castration-naïve prostate cancer.

INTRODUCTION: The last decade has seen a remarkable shift in the treatment landscape of advanced prostate cancer, none more so than in the management of metastatic castration-naïve disease. METHODS: This narrative review will examine existing and emerging evidence supporting systemic therapy use for metastatic castration-naïve prostate cancer (mCNPC) and provide guidance on the selection of these agents with respect to optimising patient outcomes. RESULTS: The addition of either docetaxel (chemohormonal approach) or an AR pathway inhibitor (abiraterone, enzalutamide or apalutamide) is a reasonable standard of care option for men commencing long-term ADT for mCNPC. While the issue of disease volume as a predictive biomarker for docetaxel benefit has previously been debated, recent data support consideration of upfront docetaxel in all patients, regardless of metastatic burden. Decisions regarding systemic treatment for men with mCNPC should be based on comprehensive consideration of disease, patient and logistical factors. Multiple novel therapeutics for mCNPC are currently under active investigation. CONCLUSION: The introduction of potent systemic therapy earlier in the mCNPC disease course has resulted in dramatic improvements in clinical outcomes for patients. As the management of mCNPC continues to evolve, the future remains promising, with the expectation of ongoing improvements to patient outcomes and quality of life.

Benefit from cytoreductive nephrectomy and the prognostic role of neutrophil-to-lymphocyte ratio in patients with metastatic renal cell carcinoma.

BACKGROUND: The role of cytoreductive nephrectomy (CN) for metastatic renal cell carcinoma (mRCC) in the era of targeted therapies is currently undefined. In recent years, neutrophil-to-lymphocyte ratio (NLR) has emerged as a prognostic marker in several cancers, including mRCC. In this multicentre retrospective study, we aim to assess the impact of CN in mRCC and the value of NLR in risk stratification and patient selection. METHODS: Retrospective data from patients with de novo mRCC from four large Australian hospitals were collected. Survival analyses were performed using the Kaplan-Meier method and compared using the log-rank test. Multivariate analyses were performed using the Cox proportional hazards method. RESULTS: Our study identified 91 de novo mRCC patients. Patients who underwent CN (n = 46, 51%) were more likely to be younger (59.0 years vs 64.6 years, P = 0.019) and to have received systemic therapy (91% vs 76%, P = 0.043). Median overall survival (mOS) was significantly improved in patients who underwent CN (23.0 months vs 10.9 months, hazard ratios (HR) 0.33, 95% confidence interval (CI) 0.20-0.55, P < 0.0001). Patients with NLR ≥ 5 also had inferior mOS (6.2 months vs 16.7 months, HR 1.94, 95% CI 1.14-3.29, P = 0.014). CN was associated with substantially improved survival in patients with both NLR < 5 (mOS 31.1 months vs 7.0 months, HR 0.41, 95% CI, 0.18-0.64, P = 0.0009) and NLR ≥ 5 (mOS 10.9 months vs 2.3 months, HR 0.33, 95% CI, 0.11-0.69, P = 0.009). Significant survival benefits associated with CN were maintained in multivariate analyses (HR 0.39, 95% CI 0.22-0.70, P = 0.0014). CONCLUSIONS: CN is associated with significantly improved overall survival in de novo mRCC. The incremental survival benefit associated with CN was seen irrespective of NLR.

A prognostic index model for predicting overall survival in patients with metastatic castration-resistant prostate cancer treated with abiraterone acetate after docetaxel.

BACKGROUND: Few prognostic models for overall survival (OS) are available for patients with metastatic castration-resistant prostate cancer (mCRPC) treated with recently approved agents. We developed a prognostic index model using readily available clinical and laboratory factors from a phase III trial of abiraterone acetate (hereafter abiraterone) in combination with prednisone in post-docetaxel mCRPC. PATIENTS AND METHODS: Baseline data were available from 762 patients treated with abiraterone-prednisone. Factors were assessed for association with OS through a univariate Cox model and used in a multivariate Cox model with a stepwise procedure to identify those of significance. Data were validated using an independent, external, population-based cohort. RESULTS: Six risk factors individually associated with poor prognosis were included in the final model: lactate dehydrogenase > upper limit of normal (ULN) [hazard ratio (HR) = 2.31], Eastern Cooperative Oncology Group performance status of 2 (HR = 2.19), presence of liver metastases (HR = 2.00), albumin ≤4 g/dl (HR = 1.54), alkaline phosphatase > ULN (HR = 1.38) and time from start of initial androgen-deprivation therapy to start of treatment ≤36 months (HR = 1.30). Patients were categorized into good (n = 369, 46%), intermediate (n = 321, 40%) and poor (n = 107, 13%) prognosis groups based on the number of risk factors and relative HRs. The C-index was 0.70 ± 0.014. The model was validated by the external dataset (n = 286). CONCLUSION: This analysis identified six factors used to model survival in mCRPC and categorized patients into three distinct risk groups. Prognostic stratification with this model could assist clinical practice decisions for follow-up and monitoring, and may aid in clinical trial design. TRIAL REGISTRATION NUMBERS: NCT00638690.

Could Nef and Vpr proteins contribute to disease progression by promoting depletion of bystander cells and prolonged survival of HIV-infected cells?

A growing body of literature suggests that the HIV accessory proteins Nef and Vpr could be involved in depletion of CD4(+) and non-CD4(+) cells and tissue atrophy, and in delaying the death of HIV-infected cells. Cell depletion is likely to be predominantly a bystander effect because the number of cells dying far outnumbers HIV-infected cells and is not confined to CD4(+) cells. The myristylated N-terminal region of Nef has severe membrane disordering properties, and when present in the extracellular medium causes rapid lysis in vitro of a wide range of CD4(+) and non-CD4(+) cells, suggesting a role for extracellular Nef in the depletion of bystander cells. A direct role for HIV-1 Nef in cytopathicity is supported by studies in HIV-infected Hu Liv/Thy SCID mice, in transgenic mice expressing nef gene alone, and in rhesus macaques infected with SIV/HIV chimeric virus containing HIV-1 nef. The N-terminal region of Nef has been directly implicated in development of simian AIDS. Extracellular Vpr and C-terminal fragments of Vpr cause membrane permeabilization and apoptosis of a wide range of CD4(+) and non-CD4(+) cells, and could also contribute to depletion of bystander cells. A direct in vivo role for Vpr in thymocyte depletion, thymic atrophy, and nephropathy is suggested in studies with vpr transgenic mice. Intracellular Nef and Vpr could help HIV-infected cells evade cell death by inhibiting apoptosis of infected cells and by avoiding virus-specific CTL response. Nef and Vpr are potential targets for therapeutic intervention and vaccine development, and strategies that prevent the death of bystander cells while promoting the early death of HIV-infected cells could arrest or retard progression to AIDS.

Structural requirements for the cytotoxicity of the N-terminal region of HIV type 1 Nef.

We have found that the hemolytic and cytotoxic activities of myristoylated Nef N-terminal peptides require a net positive charge in the first seven amino residues of the sequence. The activities are considerably less dependent on the secondary structure of the peptides. Film balance studies showed that both active and inactive peptides interacted with neutral phospholipid monolayers, suggesting that binding to neutral lipids was not a sufficient condition for lytic activity. It was also found that nonmyristoylated N-terminal peptide did not interact to the same extent with the monolayer, indicating that myristoylation was essential for lipid interaction. It is considered that the positively charged residues of the proximate N terminus of Nef interact with acidic lipids of biological membranes, reinforcing the weak membrane-targeting properties of the myristyl chain. Parallels are drawn between this mode of interaction with membranes and that of members of the Src family of proteins, which are also myristoylated and have positively charged residues in their proximate N termini. In particular, these proteins and Nef also have serine residues in their proximal N-terminal regions, which when phosphorylated could neutralize the positive charge and thus provide a mechanism for modulating membrane interaction.

Solution structure of peptides from HIV-1 Vpr protein that cause membrane permeabilization and growth arrest.

Vpr, one of the accessory gene products encoded by HIV-1, is a 96-residue protein with a number of functions, including targeting of the viral pre-integration complex to the nucleus and inducing growth arrest of dividing cells. We have characterized by 2D NMR the solution conformations of bioactive synthetic peptide fragments of Vpr encompassing a pair of H(F/S)RIG sequence motifs (residues 71-75 and 78-82 of HIV-1 Vpr) that cause cell membrane permeabilization and death in yeast and mammalian cells. Due to limited solubility of the peptides in water, their structures were studied in aqueous trifluoroethanol. Peptide Vpr59-86 (residues 59-86 of Vpr) formed an alpha-helix encompassing residues 60-77, with a kink in the vicinity of residue 62. The first of the repeated sequence motifs (HFRIG) participated in the well-defined alpha-helical domain whereas the second (HSRIG) lay outside the helical domain and formed a reverse turn followed by a less ordered region. On the other hand, peptides Vpr71-82 and Vpr71-96, in which the sequence motifs were located at the N-terminus, were largely unstructured under similar conditions, as judged by their C(alpha)H chemical shifts. Thus, the HFRIG and HSRIG motifs adopt alpha-helical and turn structures, respectively, when preceded by a helical structure, but are largely unstructured in isolation. The implications of these findings for interpretation of the structure-function relationships of synthetic peptides containing these motifs are discussed.

Inhibition of ribosomal subunit association and protein synthesis by oligonucleotides corresponding to defined regions of 18S rRNA and 5S rRNA.

Strong complementarity between a conserved sequence near the 3' end of 18S (16S) rRNA of the small ribosomal subunit and a conserved sequence in the 5S rRNA of the large ribosomal subunit supported the suggestion that base-paired interaction between the two RNA molecules could be responsible for the reversible association of ribosomal subunits during protein synthesis. If this were true then oligonucleotides corresponding to defined regions of the 18S and 5S rRNAs should have profound effects on the association of ribosomal subunits and protein synthesis. In this report we show that oligonucleotides, corresponding to a defined region of eukaryotic 18S rRNA, when bound to wheat embryo 60S ribosomal subunits, inhibited association with 40S ribosomal subunits and also inhibited in vitro protein synthesis. Similarly oligonucleotides corresponding to a defined region of 5S rRNA when bound to 40S ribosomal subunits also inhibited the formation of 80S ribosomes and in vitro protein synthesis. The minimum sequences responsible for the inhibition of ribosomal subunit association and in vitro protein synthesis corresponded to the 5' strand of the m2(6)A m2(6)A hairpin structure near the 3' end of 18S rRNA and nucleotides 91-100 of 5S rRNA which are complementary to each other. Sequences at identical positions of Escherichia coli 16S and 5S rRNAs are also complementary to each other.

Cytotoxic activity of the amino-terminal region of HIV type 1 Nef protein.

Myristoylated 21- and 25-residue N-terminal peptides of the Nef protein of HIV-1 lysed human erythrocytes and were cytotoxic toward a human CD4+ T cell line, CEM, and primary human peripheral blood mononuclear cells (PBMCs). The corresponding nonmyristoylated N-terminal peptides were only very weakly hemolytic and cytotoxic. A myristoylated peptide consisting of residues 31-50 of Nef was neither hemolytic nor cytotoxic. Alteration of the tryptophan residue at position 13 to a serine did not change the hemolytic and cytotoxic activity. Studies of the ultraviolet fluorescence of the tryptophan at position 5 in the peptide, using an artificial membrane system and fluorescence-quenching agents that inserted into the bilayer at different levels, suggested that myristoylation results in this residue being brought into contact with the upper hydrocarbon region of the lipid bilayer of the cell membrane. This tryptophan is flanked by a number of polar residues that would maintain it in this position, resulting in a considerable increase in disorder in the upper regions of the lipid bilayer, leading to its destabilization and to lysis. The cytotoxic activity of the myristoylated Nef fragments may, in part, explain the killing and deletion of cells, especially in lymphoid tissues, during HIV infection.

HIV-1 protein Vpr causes gross mitochondrial dysfunction in the yeast Saccharomyces cerevisiae.

The biological effects of the HIV-1 accessory protein, Vpr, have been studied in yeast expression systems. In our previous study [1], employing the pCUP1-vpr copper-inducible expression cassette, Vpr was shown to cause growth arrest and structural defects. In this study yeast constitutively expressing vpr, through elevated copy number and/or elevated transcription levels, displayed no growth arrest in fermentative growth conditions while Vpr was produced at much lower levels than in the inducible expression system. However, such cells were respiratory deficient and unable to utilise ethanol or glycerol as the sole carbon source. They exhibited gross mitochondrial dysfunction displayed in the loss of respiratory chain complex I, II, III, IV and citrate synthase activities. The effects on mitochondria required a C-terminal domain of Vpr that contains a conserved amino acid sequence motif HFRIGCRHSRIG. These results suggest that the widely observed phenomenon of 'Vpr-induced growth arrest' in human cells could be due to mitochondrial dysfunction.

Solution structure of a polypeptide from the N terminus of the HIV protein Nef.

Nef is a 27 kDa myristylated phosphoprotein expressed early in infection by HIV. The N terminus of Nef is thought to play a vital role in the functions of this protein through its interactions with membrane structures. The solution structure of a 25-residue polypeptide corresponding to the N terminus of Nef (Nef1-25) has been investigated by 1H NMR spectroscopy. In aqueous solution at pH 4.8 and 281 K, this peptide underwent conformational averaging, with Pro13 existing in cis and trans conformations in nearly equal proportions. In methanol solution, however, the peptide adopted a well-defined alpha-helical structure from residues 6 to 22, with the N- and C-terminal regions having a less ordered structure. On the basis of a comparison of chemical shifts and NOEs, it appeared that this helical structure was maintained in aqueous trifluoroethanol (50% v/v) and to a lesser extent in a solution of SDS micelles. When the N-acetyl group was replaced by either an N-myristyl or a free ammonium group, there was little effect on the three-dimensional structure of the peptide in methanol; deamidation of the C terminus also had no effect on the structure in methanol. In water, the myristylated peptide aggregated. The similarity between the sequences of Nef1-25 and melittin is reflected in the similar structures of the two molecules, although the N-terminal helix of melittin is more defined. This similarity in structure raises the possibility that Nef1-25 not only interacts with membranes but also may be capable of disrupting them and causing cell lysis. This type of interaction could contribute at least in part to the killing of bystander cells in lymphoid tissues during HIV infection.

Cytotoxicity resulting from addition of HIV-1 Nef N-terminal peptides to yeast and bacterial cells.

The Nef protein of human immunodeficiency type 1 (HIV-1) has been implicated in diverse intracellular functions; however, extracellular functions have been less studied. Nef and the N-terminus of Nef possess membrane-perturbing and fusogenic activities in artificial membranes that also cause cytotoxicity to human cells, including lymphocytes. The present study investigates the toxicity of HIV-1 Nef peptides employing yeast and bacterial cells. The N-terminal portion of Nef was found to cause cell killing in Escherichia coli and in a variety of yeast cells. This activity was enhanced by myristylation of the Nef N-terminus, a modification that did not lead to toxicity in a control peptide. Cell death in yeast was due to permeabilization of the cell membrane as determined by the propidium iodide uptake of peptide-treated cells. Extracellular Nef, or its breakdown products, may have effects similar to the Nef peptides described here and could be responsible, at least in part, for the death of cells in lymphoid tissues during AIDS. Assays using yeast or bacteria are convenient, inexpensive, and robust and should be useful in further analysis and screening of inhibitors of this activity associated with HIV-1 Nef.

Extracellular addition of a domain of HIV-1 Vpr containing the amino acid sequence motif H(S/F)RIG causes cell membrane permeabilization and death.

Vpr is a virion-associated protein of human immunodeficiency virus type 1 (HIV-1) whose function in acquired immune deficiency syndrome (AIDS) has been uncertain. We previously employed yeast as a model to examine the effects of Vpr on basic cellular functions; intracellular Vpr was shown to cause cell-growth arrest and structural defects, and these effects were caused by a region of Vpr containing the sequence HFRIGCRHSRIG. Here we show that peptides containing the H(S/F)RIG amino acid sequence motif cause death when added externally to a variety of yeast including Saccharomyces cerevisiae, Kluyveromyces lactis, Candida glabrata, Candida albicans and Schizosaccharomyces pombe. Such peptides rapidly entered the cell from the time of addition, resulting in cell death. Elevated levels of ions, particularly magnesium and calcium ions, abrogated the cytotoxic effect by preventing the Vpr peptides from entering the cells. Extracellular Vpr found in the serum, or breakdown products of extracellular Vpr, may have similar effects to the Vpr peptides described here and could explain the death of uninfected bystander cells during AIDS.

The hepatitis B virus X protein is a potent AMP kinase.

The hepatitis B virus X-protein (HBx) has been expressed in Escherichia coli both as an unfused protein and with an N-terminal hexaHis-containing fusion sequence. Both forms of HBx, after purification, displayed a potent AMP kinase activity, in which HBx phosphorylates AMP to ADP, using ATP as the exclusive phosphate donor. We also found that HBx has previously unreported GTPase and GTP-ADP nucleoside diphosphate kinase activities.

Stress- and sequence-dependent release into the culture medium of HIV-1 Nef produced in Saccharomyces cerevisiae.

We have produced human immunodeficiency virus type 1 (HIV-1) Nef (a myristylated 206-amino-acid protein) in Saccharomyces cerevisaie and shown that, while Nef is normally found as a predominantly intracellular protein, amounts up to 40 micrograms/ml of Nef are also released into the extracellular medium during stress. By electrophoretic (SDS-PAGE) analysis the extracellular Nef is indistinguishable from intracellular Nef. Conditions of stress that lead to the release of Nef include elevated levels of copper or magnesium ions or growth at elevated temperatures. This release appears to be dependent upon the N-terminal sequences of Nef, including the presence of a myristylation site. Our observations concerning Nef release in yeast suggest new ways in which the behaviour of Nef should be examined in order to gain further insights into the development of AIDS. If the release of Nef is important in the development of AIDS, our work reveals that Nef-associated symptoms may be reduced or delayed by reducing stresses, such as fevers.

A domain of human immunodeficiency virus type 1 Vpr containing repeated H(S/F)RIG amino acid motifs causes cell growth arrest and structural defects.

Vpr is a virion-associated protein of human immunodeficiency type 1 (HIV-1) whose function in acquired immunodeficiency syndrome (AIDS) has been uncertain. Employing the yeast Saccharomyces cerevisiae as a model to examine the effects of HIV-1 auxiliary proteins on basic cellular functions, we found that the vpr gene caused cell growth arrest and structural defects indicated by osmotic sensitivity and gross cell enlargement. Production of various domains by gene expression showed that this effect arose from within the carboxyl-terminal third of the Vpr protein and implicated the sequence HFRIGCRHSRIG, containing two H(S/F)RIG motifs. Electroporation with a series of peptides containing these motifs caused structural defects in yeast that resulted in osmotic sensitivity. A protein with functions relating to the yeast cytoskeleton, Sac1p [Cleves, A. E., Novick, P.J. & Bankaitis, V.A. (1989) J. Cell Biol. 109, 2939-2950], shows sequence similarity to Vpr, and Vpr's effect in yeast may be to disrupt normal Sac1p functions. The Sac1p equivalent has not yet been described in mammalian cells, but in rhabdomyosarcoma and osteosarcoma cell lines Vpr also caused gross cell enlargement and replication arrest [Levy, D.N., Fernandes, L.S., Williams, W.V. & Weiner, D.B. (1993) Cell 72, 541-550]. We note that there is a correlation between the region containing the H(S/F)RIG motifs and the pathogenicity of primate lentiviruses and we suggest that the function of Vpr may be to bring about cell growth arrest and/or cytoskeletal changes as an early step in HIV-1 infection.

Changes in the NS gene of neurovirulent strains of influenza affect splicing.

The nonstructural (NS) gene has been identified as an accessory factor in determining neurovirulence for influenza A virus. The nucleotide sequence of the NS gene of the neurovirulent variant A/NWS/33 was determined and compared with its parent, A/WS/33. Alterations in the mRNA structure of the gene were observed that serve to mask the 3' splice site. Changes in this region were shown to correlate with reduced splicing of the NS gene in neurovirulent strains.

Expression and analysis of the NS2 protein of influenza A virus.

Influenza NS2 protein was expressed in Saccharomyces cerevisiae using a copper-inducible promoter. The protein produced had a molecular weight of 13 kDa, was reactive with anti-NS2 antiserum and was localised to the yeast cell nucleus. Two-hybrid analysis identified a direct protein-protein interaction between NS2 and the M2 protein of the virus, involving the C-terminal 163 residues of M1. A filter-binding assay localised the M1 binding region to the C-terminal 70 amino acids of NS2.

Fusogenic activity of amino-terminal region of HIV type 1 Nef protein.

We have studied two isoforms of Nef, Nef-27 and Nef-25, which were produced in E. coli. Nef-25 lacked the first 18 N-terminal residues of Nef-27 and both were nonmyristylated. Nef-27 fuses small unilamellar dipalmitoyl phosphatidylcholine vesicles (SUVs), as indicated by enhanced light scattering of SUVs and lipid mixing using concentration-dependent fluorescence dequenching. Nef-27 also causes the appearance of a shifted isotropic peak in the 31P NMR spectra of these vesicles, suggesting that protein interactions induce nonlamellar lipid structures. Recombinant Nef-25, which lacks only the 18 N-terminal residues of Nef-27, does not fuse vesicles and has little effect on the 31P NMR spectra. On the other hand, synthetic peptides consisting of 18 or 21 of the N-terminal residues of Nef-27 are strongly membrane perturbing, causing vesicle fusion and inducing isotropic peaks in the 31P NMR spectrum. Endogenous fluorescence spectra of the N-terminal peptide (21 residues) with SUVs show that the N-terminal sequence of Nef may achieve these perturbing effects by inserting its hydrophobic side into the lipid bilayer. Theoretical calculations using hydrophobic moment plot analysis indicate that short-length stretches (i.e., six amino acid residues) of the N-terminal sequence may insert into the lipid bilayer as multimeric alpha helices or beta sheets. The above-described membrane activities of Nef-27, which principally reside in its N-terminal domain, may play critical role(s) in certain functional properties of the full-length protein. For example, the fusogenic activity of the N-terminal sequence may be involved in the extracellular release of Nef-27, much of which appears to be associated with small membrane vesicles. The fusion activity may also be relevant to the ability of Nef-27 to downregulate CD4 and IL-2 receptors when this protein is electroporated into cultured lymphocytes, an activity not possessed by Nef-25.

High-level secretion of correctly processed beta-lactamase from Saccharomyces cerevisiae using a high-copy-number secretion vector.

We have sought to obtain a convenient system for the high-level production of secreted proteins in yeast. With the aid of a secretion reporter cassette we examined the secretion of beta-lactamase (Bla) as a model protein and found the highest expression in Saccharomyces cerevisiae using a high-copy-number plasmid. We further developed the high-copy-number plasmid introducing a secretion cassette that has a convenient cloning site coinciding with the sequence encoding the KEX2 cleavage site. Large quantities of correctly-processed product can therefore be obtained. We show that 0.3 g/l of correctly processed beta-lactamase can be obtained in fed-batch cultures without the need for selective media or significant loss of the plasmid.