Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain

<p>Abstract</p> <p>Background</p> <p>The expression of heterologous proteins in <it>Escherichia coli </it>is strongly affected by codon bias. This phenomenon occurs when the codon usage of the mRNA coding for the foreign protein differs from that of the bacterium. The ribosome pauses upon encountering a rare codon and may detach from the mRNA, thereby the yield of protein expression is reduced. Several bacterial strains have been engineered to overcome this effect. However, the increased rate of translation may lead to protein misfolding and insolubilization. In order to prove this assumption, the solubility of several recombinant proteins from plants was studied in a codon bias-adjusted <it>E. coli </it>strain.</p> <p>Results</p> <p>The expression of eight plant proteins in <it>Escherichia coli </it>BL21(DE3)-pLysS and BL21(DE3)-CodonPlus-pRIL was systematically studied. The CodonPlus strain contains extra copies of the <it>argU</it>, <it>ileY</it>, and <it>leuW </it>tRNA genes, which encode tRNAs that recognize the codons AGA/AGG, AUA and CUA, respectively (RIL codons). The level of expression and solubility of the recombinant proteins were analyzed by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting. We found that for all proteins the solubility was at least 25% in the BL21(DE3)-pLysS strain. However, when expressed in the BL21(DE3)-CodonPlus-pRIL strain, proteins having more than 5% of amino acids coded by RIL codons were localized mainly in the insoluble fraction. Also, their expression caused retarded growth and low cell yield in the codon bias-adjusted strain at all temperatures tested. On the contrary, the solubility of proteins containing less than 5% of amino acids coded by RIL codons remained unchanged in both strains and their expression caused no effect on cell growth.</p> <p>Conclusion</p> <p>Our results show that the expression of heterologous proteins coded by high RIL codon content coding sequences in a codon bias-adjusted strain is detrimental for their solubility. Our data support the hypothesis that the possible elimination of translational pauses that increase translation rate leads to protein misfolding and aggregation. This stresses the importance of strain selection according to codon content in any scheme where a large amount of biologically active product is desirable.</p

Comprehensive Overview of Bottom-up Proteomics using Mass Spectrometry

Proteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. "Shotgun proteomics" or "bottom-up proteomics" is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods to aid the novice and experienced researcher. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this work to serve as a basic resource for new practitioners in the field of shotgun or bottom-up proteomics

Structural basis for the Pr-Pfr long-range signaling mechanism of a full-length bacterial phytochrome at the atomic level

Phytochromes constitute a widespread photoreceptor family that typically interconverts between two photostates called Pr (red light–absorbing) and Pfr (far-red light–absorbing). The lack of full-length structures solved at the (near-)atomic level in both pure Pr and Pfr states leaves gaps in the structural mechanisms involved in the signal transmission pathways during the photoconversion. Here, we present the crystallographic structures of three versions from the plant pathogen Xanthomonas campestris virulence regulator XccBphP bacteriophytochrome, including two full-length proteins, in the Pr and Pfr states. The structures show a reorganization of the interaction networks within and around the chromophore-binding pocket, an α-helix/β-sheet tongue transition, and specific domain reorientations, along with interchanging kinks and breaks at the helical spine as a result of the photoswitching, which subsequently affect the quaternary assembly. These structural findings, combined with multidisciplinary studies, allow us to describe the signaling mechanism of a full-length bacterial phytochrome at the atomic level.DFG, 221545957, SFB 1078: Proteinfunktion durch ProtonierungsdynamikEC/H2020/664726/EU/EMBL Interdisciplinary, International and Intersectorial Postdocs/EI3PO

Chloroplastic Hsp100 chaperones ClpC2 and ClpD interact <it>in vitro</it> with a transit peptide only when it is located at the N-terminus of a protein

<p>Abstract</p> <p>Background</p> <p>Clp/Hsp100 chaperones are involved in protein quality control. They act as independent units or in conjunction with a proteolytic core to degrade irreversibly damaged proteins. Clp chaperones from plant chloroplasts have been also implicated in the process of precursor import, along with Hsp70 chaperones. They are thought to pull the precursors in as the transit peptides enter the organelle. How Clp chaperones identify their substrates and engage in their processing is not known. This information may lie in the position, sequence or structure of the Clp recognition motifs.</p> <p>Results</p> <p>We tested the influence of the position of the transit peptide on the interaction with two chloroplastic Clp chaperones, ClpC2 and ClpD from <it>Arabidopsis thaliana</it> (AtClpC2 and AtClpD). The transit peptide of ferredoxin-NADP⁺ reductase was fused to either the N- or C-terminal end of glutathione <it>S</it>-transferase. Another fusion with the transit peptide interleaved between two folded proteins was used to probe if AtClpC2 and AtClpD could recognize tags located in the interior of a polypeptide. We also used a mutated transit peptide that is not targeted by Hsp70 chaperones (TP1234), yet it is imported at a normal rate. The fusions were immobilized on resins and the purified recombinant chaperones were added. After a washing protocol, the amount of bound chaperone was assessed. Both AtClpC2 and AtClpD interacted with the transit peptides when they were located at the N-terminal position of a protein, but not when they were allocated to the C-terminal end or at the interior of a polypeptide.</p> <p>Conclusions</p> <p>AtClpC2 and AtClpD have a positional preference for interacting with a transit peptide. In particular, the localization of the signal sequence at the N-terminal end of a protein seems mandatory for interaction to take place. Our results have implications for the understanding of protein quality control and precursor import in chloroplasts.</p

Characterization of the accessory protein ClpT1 from Arabidopsis thaliana: oligomerization status and interaction with Hsp100 chaperones

El presente articulo se encuentra aceptado con modificaciones. Background: The caseinolytic protease (Clp) is crucial for chloroplast biogenesis and proteostasis. The Arabidopsis Clp consists of two heptameric rings (P and R rings) assembled from nine distinct subunits. Hsp100 chaperones (ClpC1/2 and ClpD) are believed to dock to the axial pores of Clp and then transfer unfolded polypeptides destined to degradation. The adaptor proteins ClpT1 and 2 attach to the protease, apparently blocking the chaperone binding sites. This competition was suggested to regulate Clp activity. Also, monomerization of ClpT1 from dimers in the stroma triggers P and R ring association. So, oligomerization status of ClpT1 seems to control the assembly of the Clp protease. Results: In this work, ClpT1 was obtained in a recombinant form and purified. In solution, it mostly consists of monomers while dimers represent a small fraction of the population. Enrichment of the dimer fraction could only be achieved by stabilization with a crosslinker reagent. We demonstrate that ClpT1 specifically interacts with the Hsp100 chaperones ClpC2 and ClpD. In addition, ClpT1 stimulates the ATPase activity of ClpD by more than 50% when both are present in a 1:1 ratio. Outside this optimal proportion, the stimulatory effect of ClpT1 on the ATPase activity of ClpD declines. Conclusions: The accessory protein ClpT1 behaves as a monomer in solution. It interacts with the chloroplastic Hsp100 chaperones ClpC2 and ClpD and tightly modulates the ATPase activity of the latter. Our results provide new experimental evidence that may contribute to revise and expand the existing models that were proposed to explain the roles of this poorly understood regulatory protein.Fil: Colombo, Clara Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Ceccarelli, Eduardo Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Rosano, German Leandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentin

Analysis of Pin1 expression in the adult zebrafish brain.

<p>Immunofluorescence analysis on brain coronal sections using Pin1 (green) or HuC/D (red) as primary antibodies. (A) and (B) dorsal telencephalon, (C) ventral diencephalon, (D) optic tectum, (E) corpus cerebelli, (F) romboencephalic ventricular zone. DiV: diencephalic ventricle, Dd: lateral zone of dorsal telencephalon, Dm: medial zone of dorsal telencephalon, GL: cerebellar granular layer, Hv: ventral zone of periventricular hypothalamus, IML: cerebellar intermediate layer, PGZ: periventricular gray zone of the optic tectum, RV: romboencephalic ventricle, TelV: telencephalic ventricle. Scale bar = 50 μm.</p

<i>pin1</i> mRNA injection affects head development in zebrafish.

<p>(A) Lateral views of live 3 dpf embryos injected with EGFP mRNA or EGFP-Pin1 mRNA (Pin1) at 1–2 cell stage. (B) Quantification of the percentage of embryos with altered head development upon injection of mRNAs coding for EGFP, EGFP-Pin1, EGFP-Pin1C109A or EGFP-WW, as indicated. Embryos with evident facial retraction, reduced mandible and/or reduced head size were scored as positive. The graph shows the average of 4 experiments with at least 50 embryos each and the standard deviation. Statistical analysis were carried out using one-way ANOVA followed by Tukey's Multiple Comparison Test: *** p˂ 0,0001. (C) Representative confocal projections of embryos microinjected with EGFP mRNA or EGFP-Pin1 mRNA, stained for apoptosis by whole-mount TUNEL assay (left panels) or for proliferation by p-H3 whole-mount immunofluorescence (right panels). (D) Quantification of apoptotic cells on heads of wild type (wt) or <i>tp53</i> ^{<i>zdf1/zdf1</i>} embryos microinjected with EGFP mRNA or EGFP-Pin1 mRNA as indicated. Statistical analyses were carried out using one-way ANOVA followed by Tukey's Multiple Comparison Test: *** p˂ 0,0001 (n = 17 for each condition). (E) Quantification of cells positive for p-H3 staining on heads of wild type embryos microinjected with EGFP mRNA or EGFP-Pin1 mRNA as indicated. Statistical analyses were carried out using unpaired t test with Welch's correction (n = 21 for each condition). (F) Morphological analysis at 3 dpf of wild type or <i>tp53</i> ^{<i>zdf1/zdf1</i>} embryos microinjected with EGFP mRNA or EGFP-Pin1 mRNA as indicated. The graph shows the average of 4 experiments with at least 50 embryos each and the standard deviation. Statistical analysis were carried out using one-way ANOVA followed by Tukey's Multiple Comparison Test: *** p˂ 0,0001.</p

Analysis of Pin1 isovariants in zebrafish extracts.

<p>(A) Representative images of two-dimensional (2D) western blot analysis of zebrafish extracts from 4:30 and 24 hpf embryos probed with anti-Pin1. Left and right panels exhibit different exposure times of the same immunoblot. (B) Left panels: magnification of the immunoblots shown in (A). Pin1 isovariants are numbered from the most basic (1) to the most acidic (4). Right panels: 2D western blot analysis of zebrafish extracts from 4:30 and 24 hpf embryos treated with λ phosphatase. pI: isoelectric point, MW: molecular weight.</p