cerevisiae-based platform for rapid production and evaluation of Eukaryotic Nutrient transporters and transceptors for biochemical studies and crystallography
A new effective S. cerevisiae expression system is developed that may be used for production of high-quality eukaryotic membrane proteins for functional and structural analysis.
A case-study evaluation of a pBR322-derived expression system, previously applied to therapeutic peptides and insulin analogs, in combination with fed-batch cultivation for recombinant production of methionine aminopeptidase in E. coli, evaluating the performance of this system for methionine aminopeptidase as a stress-sensitive model protein under the tested conditions without a side-by-side comparison with alternative expression systems.
G. Kuznetsov, Marina Yarovikova, E. Buslaeva et al.· Protein Expression and Purif...· 0 citations
This review summarizes recent advances in engineering key expression elements underlying heterologous protein production in K. phaffii, with particular emphasis on promoter architecture redesign, signal peptide replacement and sequence engineering, molecular chaperone co-expression, and quantitative regulation of the unfolded protein response.
Ru-Yue Han, Ruizheng Hu, An-Ran Liu et al.· Journal of Fungi· 1 citation
Membrane-embedded bacterial receptors are challenging to express and purify in soluble form, yet their isolated domains are essential tools for structural and ligand-discovery studies. Pseudomonas aeruginosa relies on the TonB-dependent heme receptor HasR for iron acquisition, a process central to its pathogenicity. Here, we report a robust strategy for the recombinant expression, purification, and biophysical characterisation of the two soluble HasR domains directly involved in heme uptake: the N-terminal plug and the Secretin/TonB short N-terminal domain. Each domain was expressed individually in E. coli and purified to homogeneity, adopting well-folded conformations as confirmed by circular dichroism, NMR spectroscopy, and mass spectrometry. We then engineered a fusion construct containing both domains and systematically evaluated multiple solubilisation tags. A GST-His dual-affinity strategy enabled efficient purification of the construct, whereas His-tag alone resulted in insoluble protein and HLT-tag fusions suffered from non-specific proteolysis. Biophysical analyses revealed that the Secretin/TonB short N-terminal domain remains stably folded within the fusion construct, while the N-terminal plug domain becomes partially disordered, a finding further supported by hydrogen/deuterium exchange mass spectrometry. Together, these results establish a generalizable workflow for producing soluble receptor domains from membrane proteins and provide validated HasR constructs suitable for downstream ligand-screening applications, including aptamer and nanobody discovery.
Federico Bosetto, Maria Zacharopoulou, M. Bycroft et al.· bioRxiv· 0 citations
A key goal of many bacterial engineering projects is to produce protein, due to their potent chemical and material functions. However, recombinant proteins are usually produced intracellularly, and secreting arbitrary proteins directly into an extracellular environment is challenging. In this work, we explored recombinant protein secretion using the curli secretion pathway in the probiotic Escherichia coli Nissle 1917 strain (EcN). We developed a Green Fluorescent Protein (GFP) secretion system by fusing GFP of the N-terminus sequence from the curli monomer protein while co-expressing the curli export machinery. We characterized and optimized our designs, obtaining ∼60 μg/mL extracellular GFP protein from plasmid pSecGFP1h. To further increase protein yield, we performed a directed evolutionary process using chemical mutagenesis and selecting for both high GFP yield and secretion of a protein toxic to E. coli. We performed several rounds of mutagenesis and selection to generate two distinct lineages, which both secreted ∼120 μg/mL extracellular GFP. We then sequenced and analyzed the genomes of strains making up the two lineages, assembling genome sequences for 8 EcN-derived strains, finding ∼50 point mutations per round of mutagenesis, and identifying the genes affected. We further assessed the capability of our evolved E. coli strains to secrete diverse proteins, including proteinaceous materials, enzymes, and therapeutic peptides. We obtained successful secretion for many proteins and found that secretion was sequence-specific, although no simple metric could predict success. The methodology we developed for microbial strain engineering and protein secretion has the potential to be generalizable to many biotechnological applications to take advantage of the abilities of proteins.
Anton Kan, S. Emani, Neel S. Joshi· ACS Synthetic Biology· 0 citations
Pichia pastoris is a widely used host for recombinant protein production because it combines the advantages of microbial cultivation with eukaryotic protein folding and secretion. However, secretion efficiency is often limited by the folding capacity of the endoplasmic reticulum (ER), where recombinant proteins must be translocated, folded, and processed prior to export. When ER folding capacity is exceeded, proteins may be retained, degraded, or secreted in non-native conformations, reducing both yield and product quality. Chaperone engineering and codon optimization represent two promising strategies to address these limitations. Here, we generated stable Pichia strains expressing four model secreted proteins (human serum albumin, interleukin-2, thaumatin-I, and thaumatin-II) using either conventional codon optimization or Epi-MAX codon engineering, which adapts transgene codon usage to stress-responsive translational programs. We also engineered strains containing an additional chromosomal copy of either the ER Hsp70 chaperone Kar2 or protein disulfide isomerase (Pdi1). To assess protein quality, we applied limited proteolysis mass spectrometry (LiP-MS), a structural proteomics approach that can detect subtle conformational differences to secreted proteins. Increased Pdi1 levels improved secretion of all four proteins tested, whereas Kar2 overexpression generally reduced yield. For thaumatin-II, Pdi1 enhanced secretion but promoted release of a non-native conformation, which we could correct through codon engineering. Together, these results demonstrate that maximizing recombinant protein production requires optimization of both yield and structural quality and establish complementary strategies for improving secreted protein expression in Pichia.
Protein-glutamine glutaminase (PGG) is a promising enzyme for improving the functionality of plant proteins, but its industrial production is limited by low native yields and the need for proteolytic activation. Here, we developed a standardized modular Golden Gate-based secretion platform in
Bacillus subtilis
to screen promoter-signal peptide combinations for extracellular production of
Chryseobacterium proteolyticum
PGG (
Cp
PGG) within a common construct architecture. Reporter and enzyme-based screening identified Pgrac100-amyQ* as the configuration that consistently supported robust secretion and functional
Cp
PGG production in
B. subtilis
168, where the secreted proenzyme underwent host-mediated maturation. In 3-L batch bioreactor cultivation in rich medium, this construct reached 2.9 U/mL of supernatant (SN), whereas fed-batch cultivation in defined medium revealed extracellular proteolysis as a major limitation under high-cell-density conditions. To decouple secretion from activation, the construct was transferred to the protease-deficient strain KO7-S which enabled stable accumulation of non-processed
Cp
PGG. Subsequent controlled in vitro activation with a food-grade neutral protease yielded 12.8 ± 0.8 U/mL SN. Together, these results show that efficient PGG production in
B. subtilis
requires coordinated control of secretion, extracellular stability and proenzyme activation.
•
Modular screening identified Pgrac100-amyQ* for CpPGG secretion in B. subtilis.
•
Proteolysis limited high-cell-density fed-batch production in B. subtilis 168.
•
In Bs KO7-S, secretion and activation were decoupled yielding 12.8 ± 0.8 U/mL SN.
Clàudia Lliso-Pascual, Sergi Abad, Marc Carnicer et al.· Applied Microbiology and Bio...· 0 citations
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