Recombinant expression of Chlamydia trachomatis major outer membrane protein in E. Coli outer membrane as a substrate for vaccine research

Wen, Zhiping; Boddicker, Melissa A.; Kaufhold, Robin M.; Khandelwal, Puneet; Dürr, Eberhard; Qiu, Ping; Lucas, Bob J.; Nahas, Debbie D.; Cook, James C.; Touch, Sinoeun; Skinner, Julie M.; Espeseth, Amy S.; Przysiecki, Craig T.; Zhang, Lan

doi:10.1186/s12866-016-0787-3

Cited by 18 publications

(21 citation statements)

References 40 publications

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“…Structural elucidation of MOMP has been hampered by the requirement for detergents to keep the membrane protein in solution during purification, as well as the lack of a recombinant expression system that preserves MOMP in its native conformation. Despite increasing efforts to express recombinant MOMP, 28 purification and reconstitution remain problematic, and extracting MOMP in its native form from Chlamydia preparations appears to be the optimal solution for the purpose of structure determination.…”

Section: Introductionmentioning

confidence: 99%

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation

et al. 2018

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Chlamydial major outer membrane protein (MOMP) is the major protein constituent of the bacterial pathogen Chlamydia trachomatis. Chlamydia trachomatis Serovars D–K are the leading cause of genital tract infections which can lead to infertility or ectopic pregnancies. A vaccine against Chlamydia is highly desirable but currently not available. MOMP accounts for ~ 60% of the chlamydial protein mass and is considered to be one of the lead vaccine candidates against C. trachomatis. We report on the spectroscopic analysis of C. trachomatis native MOMP Serovars D, E, F, and J as well as C. muridarum MOMP by size exclusion chromatography multi angle light scattering (SEC MALS), circular dichroism (CD) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR‐FTIR). MOMP was purified from the native bacterium grown in either adherent HeLa cells or in different suspension cell lines. Our results confirm that MOMP forms homo‐trimers in detergent micelles. The secondary structure composition of C. trachomatis MOMP was conserved across serovars, but different from composition of C. muridarum MOMP with a 13% (CD) to 18% (ATR‐FTIR) reduction in β‐sheet conformation for C. trachomatis MOMP. When Serovar E MOMP was isolated from suspension cell lines the α‐helix content increased by 7% (CD) to 13% (ATIR‐FTIR). Maintenance of a native‐like tertiary and quaternary structure in subunit vaccines is important for the generation of protective antibodies. This biophysical characterization of MOMP presented here serves, in the absence of functional assays, as a method for monitoring the structural integrity of MOMP.

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Section: Introductionmentioning

confidence: 99%

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation

et al. 2018

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“…5b). These multimeric structures are not evident in recombinant MOMP produced in traditional E. coli expression systems with the exception of the Wen et al (13) study in which MOMP is target-expressed in the E. coli outer membrane. This suggests that the environment in which MOMP sits remains critical for correct protein folding.…”

Section: Cell-free Production Of Functional and Immunogenic Mopn-mompmentioning

confidence: 96%

“…mation such as oligomer formation (12,46). It is noteworthy that in a recent study, Wen et al (13) were able to express recombinant MOMP on E. coli outer membrane that allowed for native-like oligomer formation. They further demonstrated that the oligomer formation is critical for effective immunogenicity (13).…”

Section: Cell-free Production Of Functional and Immunogenic Mopn-mompmentioning

confidence: 99%

“…MOMP is a complex protein that comprises 16 transmembrane domains, rendering it difficult to recombinantly synthesize in a correctly folded state. Recently, Wen et al (13) expressed C. trachomatis MOMP on Escherichia coli outer membrane and demonstrated improved protein folding and immunogenicity. However, in that study, they had to alter the gene sequence to achieve the targeted expression.…”

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confidence: 99%

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Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development

Felderman

Evans

et al. 2017

Journal of Biological Chemistry

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Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. infection, therefore, constitutes a significant public health threat, underscoring the need for a -specific vaccine. strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, and protein misfolding. Here, we used an -based cell-free system to express a MOMP protein from the mouse-specific species (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP-tNLP). The cell-free expressed mMOMP-tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP-tNLP complex in a 1-ml cell-free reaction. The mMOMP-tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP-tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity.

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“…Based on the combination of some the above-mentioned strategies, namely, Bcodon harmonization,^use of low copy number vectors with moderate strength, suitable leader sequences, and optimization of cell culture conditions, increased targeting to E. coli outer membrane of Chlamydia trachomatis major outer membrane protein was observed and the formation of inclusion bodies avoided (Wen et al 2016). On the other hand, prokaryotic expression vectors using the rhaB promoter which are almost completely repressed until induced can be suitable for the expression of toxic proteins (Giacalone et al 2006).…”

Section: Genetic-level Strategiesmentioning

confidence: 99%

Smoothing membrane protein structure determination by initial upstream stage improvements

Pedro

Queiroz

Passarinha

2019

Appl Microbiol Biotechnol

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Membrane proteins (MP) constitute 20-30% of all proteins encoded by the genome of various organisms and perform a wide range of essential biological functions. However, despite they represent the largest class of protein drug targets, a relatively small number high-resolution 3D structures have been obtained yet. Membrane protein biogenesis is more complex than that of the soluble proteins and its recombinant biosynthesis has been a major drawback, thus delaying their further structural characterization. Indeed, the major limitation in structure determination of MP is the low yield achieved in recombinant expression, usually coupled to low functionality, pinpointing the optimization target in recombinant MP research. Recently, the growing attention that have been dedicated to the upstream stage of MP bioprocesses allowed great advances, permitting the evolution of the number of MP solved structures. In this review, we analyse and discuss effective solutions and technical advances at the level of the upstream stage using prokaryotic and eukaryotic organisms foreseeing an increase in expression yields of correctly folded MP and that may facilitate the determination of their three-dimensional structure. A section on techniques used to protein quality control and further structure determination of MP is also included. Lastly, a critical assessment of major factors contributing for a good decision-making process related to the upstream stage of MP is presented.

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Recombinant expression of Chlamydia trachomatis major outer membrane protein in E. Coli outer membrane as a substrate for vaccine research

Cited by 18 publications

References 40 publications

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation

Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development

Smoothing membrane protein structure determination by initial upstream stage improvements

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