Soluble Diphtheria Toxin Variant, CRM 197 was Obtained in Escherichia coli at High Productivity Using SUMO Fusion and an Adjusted Expression Strategy.

BACKGROUND: CRM197, a non-toxic diphtheria toxin variant, is widely used as a polysaccharide carrier in a variety of conjugate vaccines and also exhibits antitumor activity. CRM197 commercial production is limited due to the low yield of Corynebacterium diphtheriae C7 (197) tox-. Developing an efficient method for recombinant CRM197 production reduces production costs and is critical for expanding the application coverage of related medical products and basic research. Escherichia coli is a frequently used host for heterologous protein synthesis. However, the primary limitation of this system is the inclusion body formation and the low yield of active protein recovery. OBJECTIVE: As a result, we attempted to produce CRM197 in the soluble form in E. coli using a small ubiquitin-related modifier (SUMO) tag fusion and an expression strategy optimized for protein production. METHODS: CRM197 was expressed intracellularly in E. coli BL21 (DE3) with its N-terminus fused to a SUMO tag preceded by a histidine tag (HSCRM197). To improve the solubility of HSCRM197 in E. coli, a response surface method (RSM) experimental design was used based on three factors: expression temperature, inducer concentration, and sorbitol inclusion in the culture medium. Metal affinity chromatography was used to purify HSCRM197, and the SUMO tag was removed using the SUMO protease's catalytic domain. After adsorbing the SUMO tag on a Ni-NTA column, CRM197 was obtained. DNA degradation activity was determined for both HSCRM197 and CRM197. RESULTS: When HSCRM197 was expressed in E. coli under common expression conditions (37ºC, 1000 µM inducer), 15.4% of the protein was found in the cellular soluble fraction. However, when the RSM-derived expression conditions were used (30ºC, 510 µM inducer, and 200 mM sorbitol), the obtained HSCRM197 was almost completely soluble (96.5% solubility), and the system productivity was 32.67 µg ml-1 h-1. HSCRM197 and CRM197 both exhibited nuclease activity. However, the activity of CRM197 was greater than that of HSCRM197. CONCLUSION: These findings established the utility of the method developed in this study to produce CRM197 for medical applications.

Chaperones Promote Remarkable Solubilization of Salmonella enterica serovar Enteritidis Flagellin Expressed in Escherichia coli.

BACKGROUND: Flagellin of Salmonella enterica serovar Enteritidis (SEF) stimulates immune responses to both itself and coapplied antigens. It is therefore used in vaccine development and immunotherapy. Removal of pathogenic S. enterica ser. Enteritidis from SEF production process is advantageous due to the process safety improvement. The protein solubility analysis using SDS-PAGE indicated that 53.49% of SEF expressed in Escherichia coli formed inclusion bodies. However, the protein recovery from inclusion bodies requires a complex process with a low yield. OBJECTIVE: We thus aim to study possibility of enhancing SEF expression in E. coli in soluble form using chemical and molecular chaperones. METHODS: Chemical chaperones including arginine, sorbitol, trehalose, sodium chloride and benzyl alcohol were used as cultivation medium additives during SEF expression. SEF solubilization by coexpression of molecular chaperones DnaK, DnaJ, and GrpE was also investigated. RESULTS: All of the chemical chaperones were effective in improving SEF solubility. However, sorbitol showed the most profound effect. SEF solubilization by molecular chaperones was slightly better than that using sorbitol and this approach enhanced noticeably SEF soluble concentration and SEF solubility percentage to almost two folds and 96.37% respectively. Results of limited proteolysis assay and native PAGE indicated similar conformational states and proper folding for SEF obtained without using chaperones and for those obtained using sorbitol and the molecular chaperones. However, the molecular chaperones based system was less costly than the sorbitol based system. CONCLUSION: The coexpression of molecular chaperones was then considered as the most appropriate approach for soluble SEF production. Therefore, SEF production for medical purposes is expected to be facilitated.

Recent approaches in vaccine development against Streptococcus pneumoniae.

Streptococcus pneumoniae is a major cause of morbidity and mortality among children under 5 years of age worldwide. Vaccines have long been used for protection against pneumococcal infections. Capsular polysaccharides of pneumococci are main antigenic components of these vaccines. However, pneumococcal polysaccharide-based vaccines are not able to elicit appropriate immunological responses in young children and cannot induce the immune memory. Thus, pneumococcal conjugate vaccines were developed through chemical coupling of an immunogenic carrier protein to the capsule. The currently available pneumococcal conjugate vaccines elicited protection against the bacterium efficiently. However, these vaccines are expensive to manufacture and have limited serotype coverage. In this mini-review, therefore, we describe approaches attempted by researchers to circumvent the shortcomings of the conjugate vaccines including specifying appropriate cultivation conditions for the production of S. pneumoniae capsular antigens, development of suitable expression systems for the frequently used carrier protein in the conjugate vaccines (cross-reacting material 197), construction of protein-based vaccines, whole-cell vaccines, DNA vaccines, and using antigen delivery vehicles. Future trends in this field are also discussed.

Utilizing bacterial flagellins against infectious diseases and cancers.

The flagellum is the organelle providing motility to bacterial cells and its activity is coupled to the cellular chemotaxis machinery. The flagellar filament is the largest portion of the flagellum, which consists of repeating subunits of the protein flagellin. Receptors of the innate immune system including Toll like receptor 5, ICE protease activating factor, and neuronal apoptosis inhibitory protein 5 signal in response to bacterial flagellins. In addition to inducing innate immune responses, bacterial flagellins mediate the development of adaptive immune responses to both flagellins and coadministered antigens. Therefore, these proteins have intensively been investigated for the vaccine development and the immunotherapy. This review describes the utilization of bacterial flagellins for the construction of vaccines against infectious diseases and cancer immunotherapy. Furthermore, the key factors affecting the performance of these systems are highlighted.

Development of vaccine delivery vehicles based on lactic acid bacteria.

Live recombinant bacteria represent attractive antigen delivery systems able to induce both mucosal and systemic immune responses against heterologous antigens. The first live recombinant bacterial vectors developed were derived from attenuated pathogenic microorganisms. In addition to the difficulties often encountered in the construction of stable attenuated mutants of pathogenic organisms, attenuated pathogens may retain a residual virulence level that renders them unsuitable for the vaccination of partially immunocompetent individuals such as infants, the elderly or immunocompromised patients. As an alternative to this strategy, non-pathogenic food-grade lactic acid bacteria (LAB) maybe used as live antigen carriers. This article reviews LAB vaccines constructed using antigens other than tetanus toxin fragment C, against bacterial, viral, and parasitic infective agents, for which protection studies have been performed. The antigens utilized for the development of LAB vaccines are briefly described, along with the efficiency of these systems in protection studies. Moreover, the key factors affecting the performance of these systems are highlighted.

New strategy for enhancement of microbial viability in simulated gastric conditions based on display of starch-binding domain on cell surface.

The C-terminal region of the peptidoglycan hydrolase (CPH) of Lactococcus lactis IL1403 fused to the linker region and the starch-binding domain (SBD) of the *-amylase of Streptococcus bovis 148 was produced intracellularly in Escherichia coli. The fusion protein (CPH-SBD) was able to bind to the cell surface of Lactobacillus casei NRRL B-441 and to corn starch. Therefore, adhesion of cells to corn starch was mediated by the fusion protein. At a cell density of 10(9) cfu/ml and a starch concentration of 5 mg/ml, CPH-SBD-displaying L. casei cells aggregated with corn starch, whereas the free cells of L. casei did not form any aggregates with corn starch. After incubation in simulated gastric juice (pH 3.0, 1 h), the survival percentages of free cells, amylose-coated free cells, and free cells mixed with corn starch were 0.074%, 7.2%, and 3.1% respectively. When CPH-SBD-displaying bacteria aggregated with corn starch, their survival percentage was 8% higher than that of free cells mixed with corn starch. The survival of the amylose-coated CPH-SBD-displaying L. casei cells was comparable to that of amylose-coated free cells, whereas the survival percentage of amylose-coated aggregates of CPH-SBD-displaying bacteria with corn starch was 28% higher than that of amylose-coated mixture of free cells with corn starch. These results demonstrate the potential usefulness of the cell-surface display technique for enhancement of the delivery of viable microorganisms to the intestinal tract.

Bidirectional cell-surface anchoring function of C-terminal repeat region of peptidoglycan hydrolase of Lactococcus lactis IL1403.

With the aim of constructing an efficient protein display system for lactic acid bacteria (LABs), the effect of fusion direction on the cell-surface binding activity of the C-terminal region of the peptidoglycan hydrolase (CPH) of Lactococcus lactis IL1403 was studied. CPH fused to the alpha-amylase (AMY) of Streptococcus bovis 148 either at its C-terminus (CPH-AMY) or at its N-terminus (AMY-CPH) was expressed intracellularly in Escherichia coli. This domain was able to direct binding of AMY to the surface of L. lactis ATCC 19435 in both constructs. However, the number of bound molecules per cell and the specific activity for starch digestion in the case of CPH-AMY were 3 and 14 times greater than those in the case of AMY-CPH, respectively. Of the LABs tested, L. lactis ATCC 19435 showed the highest binding capability for CPH-AMY, up to 6 x 10(4) molecules per cell, with a dissociation rate constant of 5.00 x 10(-5) s(-1). The binding of CPH-AMY to the surface of Lactobacillus delbrueckii ATCC 9649 cells was very stable with a dissociation rate constant of 6.96 x 10(-6) s(-1). The production of CPH-AMY in the soluble form increased 3-fold as a result of coexpression with a molecular chaperone, trigger factor. The results of this study suggest the usefulness of CPH as a bidirectional anchor protein for the production of cell-surface adhesive enzymes in E. coli. Furthermore, the importance of the fusion direction of CPH in determining cell-surface binding and enzymatic activities was shown.

Expression of C-terminal repeat region of peptidoglycan hydrolase of Lactococcus lactis IL1403 in methylotrophic yeast Pichia pastoris.

The C-terminal region of the peptidoglycan hydrolase (CPH) of Lactococcus lactis IL1403 produced intracellularly in Escherichia coli was able to attach to the surface of cells of Lactobacillus casei NRRL B-441, Bacillus subtilis 168, E. coli XL1-blue and Saccharomyces cerevisiae IFO0216. Therefore, this domain is a suitable fusion partner for the adhesion of proteins to cell surfaces. The production of cell-surface adhesive proteins using this domain in Pichia pastoris is particularly attractive, because this organism has better capability to allow the correct folding of the recombinant proteins than prokaryotic hosts. However, when this domain is produced in this yeast, its cell-surface binding activity may be limited by glycosylation. In this study, therefore, we constructed a CPH mutant (CPHM) devoid of the potential N-glycosylation sites by site-directed mutagenesis. CPHM was successfully expressed extracellularly in P. pastoris (GS115) using the methanol inducible AOX1 promoter with an alpha-mating factor signal sequence, whereas the native CPH was not produced in this host. Western blot analysis revealed that the apparent molecular size of CPHM was 18 kDa greater than that of CPH produced in E. coli (32 kDa), which is attributed to O-glycosylation. However, CPHM produced in P. pastoris was capable of binding to the cell surfaces despite its modification by the yeast, and its dissociation rate constant from the surface of L. casei NRRL B-441 cells was 3.5-fold lower than that of CPH produced in E. coli. These results demonstrate the applicability of the constructed domain (CPHM) for the production of cell-surface adhesive proteins in P. pastoris.