“…Whole cell lysates were prepared by washing the first pellet in PBS followed by centrifugation and resuspension of the pellet directly in 300 μL of SDS-PAGE loading buffer and heating for 10–15 min at 95°C. The soluble and insoluble fractions were prepared as described previously [12]. To ensure that an equivalent number of cells was analyzed, culture volumes were all normalized to the same A 600 .…”
Section: Methodsmentioning
confidence: 99%
“…Absorbance at 486 nm was measured on a Molecular Device SpectraMax 190 spectrophotometer and initial rates were determined for the initial linear absorbance change [13, 14]. 70S Ribosomes were isolated according to standard methods as described elsewhere [12]. Total RNA was extracted from cells using an RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions and transcript abundance was determined by qRT-PCR as described elsewhere [15].…”
Section: Methodsmentioning
confidence: 99%
“…FACS-based screening of bacterial cell libraries was performed as described previously [11]. β-Galactosidase (β-gal) activity was determined using the standard Miller assay and scFv13-R4 activity was determined by standard ELISA with β-gal as antigen [12, 16]. Proteins were resolved by SDS-PAGE using 12% Tris-HCl gels and immunoblotted according to standard protocols.…”
The folding of many cellular proteins occurs co-translationally immediately outside the ribosome exit tunnel, where ribosomal proteins and other associated factors coordinate the synthesis and folding of newly translated polypeptides. Here, we show that the large subunit protein L29, which forms part of the exit tunnel in Escherichia coli, is required for the productive synthesis of an array of structurally diverse recombinant proteins including the green fluorescent protein (GFP) and an intracellular single-chain Fv antibody. Surprisingly, the corresponding mRNA transcript level of these proteins was markedly less abundant in cells lacking L29, suggesting an unexpected regulatory mechanism that links defects in the exit tunnel to the expression of genetic information. To further highlight the importance of L29 in maintaining protein expression, we used mutagenesis and selection to obtain L29 variants that enhanced GFP expression. Overall, our results suggest that the ribosomal exit tunnel proteins may be key targets for optimizing the overproduction of active, structurally complex recombinant proteins in bacterial cells.
“…Whole cell lysates were prepared by washing the first pellet in PBS followed by centrifugation and resuspension of the pellet directly in 300 μL of SDS-PAGE loading buffer and heating for 10–15 min at 95°C. The soluble and insoluble fractions were prepared as described previously [12]. To ensure that an equivalent number of cells was analyzed, culture volumes were all normalized to the same A 600 .…”
Section: Methodsmentioning
confidence: 99%
“…Absorbance at 486 nm was measured on a Molecular Device SpectraMax 190 spectrophotometer and initial rates were determined for the initial linear absorbance change [13, 14]. 70S Ribosomes were isolated according to standard methods as described elsewhere [12]. Total RNA was extracted from cells using an RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions and transcript abundance was determined by qRT-PCR as described elsewhere [15].…”
Section: Methodsmentioning
confidence: 99%
“…FACS-based screening of bacterial cell libraries was performed as described previously [11]. β-Galactosidase (β-gal) activity was determined using the standard Miller assay and scFv13-R4 activity was determined by standard ELISA with β-gal as antigen [12, 16]. Proteins were resolved by SDS-PAGE using 12% Tris-HCl gels and immunoblotted according to standard protocols.…”
The folding of many cellular proteins occurs co-translationally immediately outside the ribosome exit tunnel, where ribosomal proteins and other associated factors coordinate the synthesis and folding of newly translated polypeptides. Here, we show that the large subunit protein L29, which forms part of the exit tunnel in Escherichia coli, is required for the productive synthesis of an array of structurally diverse recombinant proteins including the green fluorescent protein (GFP) and an intracellular single-chain Fv antibody. Surprisingly, the corresponding mRNA transcript level of these proteins was markedly less abundant in cells lacking L29, suggesting an unexpected regulatory mechanism that links defects in the exit tunnel to the expression of genetic information. To further highlight the importance of L29 in maintaining protein expression, we used mutagenesis and selection to obtain L29 variants that enhanced GFP expression. Overall, our results suggest that the ribosomal exit tunnel proteins may be key targets for optimizing the overproduction of active, structurally complex recombinant proteins in bacterial cells.
“…The first part of this sequence corresponds to a stretch of 88 residues from the bacteriophage M13 protein 3 (p3 spacer), previously utilized as spacer element in RD selections [19]. This sequence was linked to a second element corresponding to a 21-residue stretch (aa 175 to 195) of the E. coli SecM, earlier reported to interact with the ribosome tunnel and capable of providing stable peptide–ribosome–mRNA ternary complexes even when followed by a downstream in-frame termination codon [27–30]. Here, the natural stop codon in the SecM gene was, however, replaced by an AGA triplet, corresponding to a rare arginine codon in E. coli to possibly further avoid disruption of ternary complexes during selection.Fig.…”
Affinity reagents recognizing constant parts of antibody molecules are invaluable tools in immunotechnology applications, including purification, immobilization, and detection of immunoglobulins. In this article, murine IgG1, the primary isotype of monoclonal antibodies (mAbs) was used as target for selection of novel binders from a combinatorial ribosome display (RD) library of 1011 affibody molecules. Four rounds of selection using three different mouse IgG1 mAbs as alternating targets resulted in the identification of binders with broad mIgG1 recognition and dissociation constants (KD) in the low nanomolar to low micromolar range. For one of the binders, denoted Zmab25, competition in binding to full length mIgG1 by a streptococcal protein G (SPG) fragment and selective affinity capture of mouse IgG1 Fab fragments after papain cleavage of a full mAb suggest that an epitope functionally overlapping with the SPG-binding site in the CH1 domain of mouse IgG1 had been addressed. Interestingly, biosensor-based binding experiments showed that neither human IgG1 nor bovine Ig, the latter present in fetal bovine serum (FBS) was recognized by Zmab25. This selective binding profile towards murine IgG1 was successfully exploited in species selective recovery of two different mouse mAbs from complex samples containing FBS, resembling a hybridoma culture supernatant.
“…Both eukaryotic and prokaryotic ribosome display systems with different modifications and distinctive DNA recovery procedures have been published [4,[42][43][44]. Recently, an intracellular ribosome display method has been developed using E. coli SecM translation arrest mechanism to generate PRM complexes inside living cells [45].…”
Protein production is one of the key steps in biotechnology and functional proteomics. Expression of proteins in heterologous hosts (such as in E. coli) is generally lengthy and costly. Cell-free protein synthesis is thus emerging as an attractive alternative. In addition to the simplicity and speed for protein production, cell-free expression allows generation of functional proteins that are difficult to produce by in vivo systems. Recent exploitation of cell-free systems enables novel development of technologies for rapid discovery of proteins with desirable properties from very large libraries. This article reviews the recent development in cell-free systems and their application in the large scale protein analysis.
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