A novel method for removing contaminant Hsp70 molecular chaperones from recombinant proteins

The production of recombinant proteins in bacteria has increased significantly in recent years, becoming a common tool for both research and the industrial production of proteins. One of the requirements of this methodology is to obtain the desired protein without contaminants. However, this goal cannot always be readily achieved. Multiple strategies have been developed to improve the quality of the desired protein product. Nevertheless, contamination with molecular chaperones is one of the recalcitrant problems that still affects the quality of the obtained proteins. The ability of chaperones to bind to unfolded proteins or to regions where the polypeptide chain is exposed make the removal of the contamination during purification challenging to achieve. This work aimed to develop a strategy to remove contaminating DnaK, one of the homologous Hsp70 molecular chaperones found in Escherichia coli, from purified recombinant proteins. For this purpose, we developed a methodology that captures the DnaK from the contaminating proteins by co‐incubation with a GST‐cleanser protein that has free functional binding sites for the chaperone. The cleanser protein can then be easily removed together with the captured DnaK. Here, we demonstrated the utility of our system by decontaminating a Histidine‐tagged recombinant protein in a batch process. The addition of the GST‐cleanser protein in the presence of ATP‐Mg eliminates the DnaK contamination substantially. Thus, our decontaminant strategy results versatile and straightforward and can be applied to proteins obtained with different expression and purifications systems as well as to small samples or large volume preparations.     

Comparison of cell‐based versus cell‐free mammalian systems for the production of a recombinant human bone morphogenic growth factor

The human bone morphogenetic protein‐2 (hBMP2) is a glycoprotein, which induces de novo bone formation. Here, recombinant production in stably transfected Chinese Hamster Ovary (CHO) cells is compared to transient expression in Human Embryo Kidney (HEK) cells and cell‐free synthesis in CHO cell lysates containing microsomal structures as sites of post‐translational processing. In case of the stably transfected cells, growth rates and viabilities were similar to those of the parent cells, while entry into the death phase of the culture was delayed. The maximum achievable rhBMP2 concentration in these cultures was 153 pg/mL. Up to 280 ng/mL could be produced in the transient expression system. In both cases the rhBMP‐2 was found to interact with the producer cells, which presumably contributed to the low yields. In the cell‐free system, hBMP2 yields could be increased to almost 40 μg/mL, reached within three hours. The cell‐free system thus approached productivities for the active (renatured) protein previously only recorded for bacterial hosts, while assuring comprehensive post‐translational processing.     

Production of G protein‐coupled receptors in an insect‐based cell‐free system

The biochemical analysis of human cell membrane proteins remains a challenging task due to the difficulties in producing sufficient quantities of functional protein. G protein‐coupled receptors (GPCRs) represent a main class of membrane proteins and drug targets, which are responsible for a huge number of signaling processes regulating various physiological functions in living cells. To circumvent the current bottlenecks in GPCR studies, we propose the synthesis of GPCRs in eukaryotic cell‐free systems based on extracts generated from insect (Sf21) cells. Insect cell lysates harbor the fully active translational and translocational machinery allowing posttranslational modifications, such as glycosylation and phosphorylation of de novo synthesized proteins. Here, we demonstrate the production of several GPCRs in a eukaryotic cell‐free system, performed within a short time and in a cost‐effective manner. We were able to synthesize a variety of GPCRs ranging from 40 to 133 kDa in an insect‐based cell‐free system. Moreover, we have chosen the μ opioid receptor (MOR) as a model protein to analyze the ligand binding affinities of cell‐free synthesized MOR in comparison to MOR expressed in a human cell line by “one‐point” radioligand binding experiments. Biotechnol. Bioeng. 2017;114: 2328–2338. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Production of functional bacteriorhodopsin by an Escherichia coli cell‐free protein synthesis system supplemented with steroid detergent and lipid

Cell‐free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis‐based Escherichia coli cell‐free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light‐driven proton pump bacteriorhodopsin, consisting of seven transmembrane α‐helices. The cell‐free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3–0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent‐lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.     

Cell‐free translational screening of an expression sequence tag library of Clonorchis sinensis for novel antigen discovery

The rapidly evolving cloning and sequencing technologies have enabled understanding of genomic structure of parasite genomes, opening up new ways of combatting parasite‐related diseases. To make the most of the exponentially accumulating genomic data, however, it is crucial to analyze the proteins encoded by these genomic sequences. In this study, we adopted an engineered cell‐free protein synthesis system for large‐scale expression screening of an expression sequence tag (EST) library of Clonorchis sinensis to identify potential antigens that can be used for diagnosis and treatment of clonorchiasis. To allow high‐throughput expression and identification of individual genes comprising the library, a cell‐free synthesis reaction was designed such that both the template DNA and the expressed proteins were co‐immobilized on the same microbeads, leading to microbead‐based linkage of the genotype and phenotype. This reaction configuration allowed streamlined expression, recovery, and analysis of proteins. This approach enabled us to identify 21 antigenic proteins. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:832–837, 2017     

Cell‐free protein synthesis and purification of human dopamine D2 receptor long isoform

The human dopamine D2 receptor long isoform (D2L) has significant implications in neurological and neuropsychiatric disorders such as Parkinson's disease and schizophrenia. Detailed structural knowledge of this receptor is limited owing to its highly hydrophobic nature, which leads to protein aggregation and host toxicity when expressed in cellular systems. The newly emerging field of cell‐free protein expression presents numerous advantages to overcome these challenges. This system utilizes protein synthesis machinery and exogenous DNA to synthesize functional proteins outside of intact cells. This study utilizes two different cell‐free systems for the synthesis of human dopamine D2L receptor. These include the Escherichia coli lysate‐based system and the wheat‐germ lysate‐based system. The bacterial cell‐free method used pET 100/D‐TOPO vector to synthesize hexa‐histidine‐tagged D2L receptor using a dialysis bag system; the resulting protein was purified using nickel‐nitrilotriacetic acid affinity resin. The wheat germ system used pEU–glutathione‐S‐transferase (GST) vector to synthesize GST‐tagged D2L receptor using a bilayer translation method; the resulting protein was purified using a GST affinity resin. The presence and binding capacity of the synthesized D2L receptor was confirmed by immunoblotting and radioligand competition assays, respectively. Additionally, in‐gel protein sequencing via Nano LC‐MS/MS was used to confirm protein synthesis via the wheat germ system. The results showed both systems to synthesize microgram quantities of the receptor. Improved expression of this highly challenging protein can improve research and understanding of the human dopamine D2L receptor. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:601–608, 2013     

Synthesis of membrane proteins in eukaryotic cell‐free systems

Cell‐free protein synthesis (CFPS) is a valuable method for the fast expression of difficult‐to‐express proteins as well as posttranslationally modified proteins. Since cell‐free systems circumvent possible cytotoxic effects caused by protein overexpression in living cells, they significantly enlarge the scale and variety of proteins that can be characterized. We demonstrate the high potential of eukaryotic CFPS to express various types of membrane proteins covering a broad range of structurally and functionally diverse proteins. Our eukaryotic cell‐free translation systems are capable to provide high molecular weight membrane proteins, fluorescent‐labeled membrane proteins, as well as posttranslationally modified proteins for further downstream analysis.     

Development of a full‐length human protein production pipeline

There are many proteomic applications that require large collections of purified protein, but parallel production of large numbers of different proteins remains a very challenging task. To help meet the needs of the scientific community, we have developed a human protein production pipeline. Using high‐throughput (HT) methods, we transferred the genes of 31 full‐length proteins into three expression vectors, and expressed the collection as N‐terminal HaloTag fusion proteins in Escherichia coli and two commercial cell‐free (CF) systems, wheat germ extract (WGE) and HeLa cell extract (HCE). Expression was assessed by labeling the fusion proteins specifically and covalently with a fluorescent HaloTag ligand and detecting its fluorescence on a LabChip^® GX microfluidic capillary gel electrophoresis instrument. This automated, HT assay provided both qualitative and quantitative assessment of recombinant protein. E. coli was only capable of expressing 20% of the test collection in the supernatant fraction with ≥20 μg yields, whereas CF systems had ≥83% success rates. We purified expressed proteins using an automated HaloTag purification method. We purified 20, 33, and 42% of the test collection from E. coli, WGE, and HCE, respectively, with yields ≥1 μg and ≥90% purity. Based on these observations, we have developed a triage strategy for producing full‐length human proteins in these three expression systems.     

Possible target‐related proteins and signal network of bufalin in A549 cells suggested by both iTRAQ‐based and label‐free proteomic analysis

Bufalin (BF) exhibited antiproliferation and antimigration effects on human A549 lung cancer cells. To search its target‐related proteins, protein expression profiles of BF‐treated and control cells were compared using two quantitative proteomic methods, iTRAQ‐based and label‐free proteomic analysis. A total of 5428 proteins were identified in iTRAQ‐based analysis while 6632 proteins were identified in label‐free analysis. The number of common identified proteins of both methods was 4799 proteins. By application of 1.20‐fold for upregulated and 0.83‐fold for downregulated cutoff values, 273 and 802 differentially expressed proteins were found in iTRAQ‐based and label‐free analysis, respectively. The number of common differentially expressed proteins of both methods was 45 proteins. Results of bioinformational analysis using Metacore^TM showed that the two proteomic methods were complementary and both suggested the involvement of oxidative stress and regulation of gene expression in the effects of BF, and fibronectin‐related pathway was suggested to be an important pathway affected by BF. Western blotting assay results confirmed BF‐induced change in levels of fibronectin and other related proteins. Overexpression of fibronectin by plasmid transfection ameliorated antimigration effects of BF. Results of the present study provided information about possible target‐related proteins and signal network of BF.     

A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell‐free protein synthesis systems

Cell‐free protein synthesis (CFPS) systems allow for robust protein expression with easy manipulation of conditions to improve protein yield and folding. Recent technological developments have significantly increased the productivity and reduced the operating costs of CFPS systems, such that they can compete with conventional in vivo protein production platforms, while also offering new routes for the discovery and production of biotherapeutics. As cell‐free systems have evolved, productivity increases have commonly been obtained by addition of components to previously designed reaction mixtures without careful re‐examination of the essentiality of reagents from previous generations. Here we present a systematic sensitivity analysis of the components in a conventional Escherichia coli CFPS reaction mixture to evaluate their optimal concentrations for production of the immunoglobulin G trastuzumab. We identify eight changes to the system, which result in optimal expression of trastuzumab. We find that doubling the potassium glutamate concentration, while entirely eliminating pyruvate, coenzyme A, NAD, total tRNA, folinic acid, putrescine and ammonium glutamate, results in a highly productive cell‐free system with a 95% reduction in reagent costs (excluding cell‐extract, plasmid, and T7 RNA polymerase made in‐house). A larger panel of other proteins was also tested and all show equivalent or improved yields with our simplified system. Furthermore, we demonstrate that all of the reagents for CFPS can be combined in a single freeze‐thaw stable master mix to improve reliability and ease of use. These improvements are important for the application of the CFPS system in fields such as protein engineering, high‐throughput screening, and biotherapeutics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:823–831, 2015     

Translation‐coupled protein folding assay using a protease to monitor the folding status

Protein folding is an essential prerequisite for proteins to execute nearly all cellular functions. There is a growing demand for a simple and robust method to investigate protein folding on a large‐scale under the same conditions. We previously developed a global folding assay system, in which proteins translated using an Escherichia coli‐based cell‐free translation system are centrifuged to quantitate the supernatant fractions. Although the assay is based on the assumption that the supernatants contain the folded native states, the supernatants also include nonnative unstructured proteins. In general, proteases recognize and degrade unstructured proteins, and thus we used a protease to digest the unstructured regions to monitor the folding status. The addition of Lon protease during the translation of proteins unmasked subfractions, not only in the soluble fractions but also in the aggregation‐prone fractions. We translated ～90 E. coli proteins in the protease‐inclusion assay, in the absence and presence of chaperones. The folding assay, which sheds light on the molecular mechanisms underlying the aggregate formation and the chaperone effects, can be applied to a large‐scale analysis.     

Coexpression of molecular chaperones to enhance functional expression of anti-BNP scFv in the cytoplasm of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the detection of B-type natriuretic peptide

Molecular chaperones are a ubiquitous family of cellular proteins that mediate the correct folding of other target polypeptides. In our previous study, the recombinant anti-BNP scFv, which has promising applications for diagnostic, prognostic, and therapeutic monitoring of heart failure, was expressed in the cytoplasm of Escherichia coli. However, when the anti-BNP scFv was expressed, 73.4% of expressed antibodies formed insoluble inclusion bodies. In this study, molecular chaperones were coexpressed with anti-BNP scFv with the goal of improving the production of functional anti-BNP in the cytoplasm of E. coli. Five sets of molecular chaperones were assessed for their effects on the production of active anti-BNP scFv. These sets included the following: trigger factor (TF); groES/groEL; groES/groEL/TF; dnaK/dnaJ/grpE; groES/groEL/dnaK/dnaJ/grpE. Of these chaperones, the coexpression of anti-BNP scFv with the groES/groEL chaperones encoded in plasmid pGro7 exhibited the most efficient functional expression of anti-BNP scFv as an active form. Coexpressed with the groES/groEL chaperones, 64.9% of the total anti-BNP scFv was produced in soluble form, which is 2.4 times higher scFv than that of anti-BNP scFv expressed without molecular chaperones, and the relative binding activity was 1.5-fold higher. The optimal concentration of l-arabinose required for induction of the groES/groEL chaperone set was determined to be 1.0 mM and relative binding activity was 3.5 times higher compared with that of no induction with l-arabinose. In addition, soluble anti-BNP scFv was increased from 11.5 to 31.4 μg/ml with optimized inducer concentration (1.0 mM l-arabinose) for the coexpression of the groES/groEL chaperones. These results demonstrate that the functional expression of anti-BNP scFv can be improved by coexpression of molecular chaperones, as molecular chaperones can identify and help to refold improperly folded anti-BNP scFv.     

Proteomic understanding of intracellular responses of recombinant chinese hamster ovary cells adapted to grow in serum‐free suspension culture

To understand the intracellular responses in recombinant Chinese hamster ovary (rCHO) cells adapted to grow in serum‐free suspension culture, a proteomic approach was employed. After rCHO cells producing erythropoietin were adapted to grow in suspension culture with the two different serum‐free media (SFM4CHO? and SF‐L1), proteome analyses were carried out using 2‐D PAGE and based on spot intensities, 58 high‐intensity protein spots were selected. Of the 58 protein spots, which represented 34 different kinds of proteins, 55 were identified by MALDI‐TOF‐MS, and MS/MS. Compared with the results in serum‐containing medium, six proteins, four de novo synthesis of nucleotides‐related proteins (dihydrolipoamide S‐acetyltransferase, transaldolase, inosine‐5′‐monophosphate dehydrogenase 2, and lymphoid‐restricted membrane protein) and two molecular chaperones (heat shock protein 70 kDa and 60 kDa [HSC70, HSP60]) were significantly increased in SFM4CHO?. From the results of proteomic analysis, HSP60 and HSC70, which were increased in both SFM, were selected as candidate proteins for engineering and rCHO cell lines overexpressing these genes were constructed. Cells overexpressing HSP60 and/or HSC70 showed 10–15% enhanced cell concentration during serum‐free adaptation and 15–33% reduction in adaptation time. Taken together, identification of differentially expressed proteins in rCHO cells by a proteomic study can provide insights into understanding the intracellular events and clues to find candidate genes for cell engineering for improved performance of rCHO cells during adaptation to serum‐free suspension culture. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Strategy for comprehensive identification of human N‐myristoylated proteins using an insect cell‐free protein synthesis system

To establish a strategy for the comprehensive identification of human N‐myristoylated proteins, the susceptibility of human cDNA clones to protein N‐myristoylation was evaluated by metabolic labeling and MS analyses of proteins expressed in an insect cell‐free protein synthesis system. One‐hundred‐and‐forty‐one cDNA clones with N‐terminal Met‐Gly motifs were selected as potential candidates from ～2000 Kazusa ORFeome project human cDNA clones, and their susceptibility to protein N‐myristoylation was evaluated using fusion proteins, in which the N‐terminal ten amino acid residues were fused to an epitope‐tagged model protein. As a result, the products of 29 out of 141 cDNA clones were found to be effectively N‐myristoylated. The metabolic labeling experiments both in an insect cell‐free protein synthesis system and in the transfected COS‐1 cells using full‐length cDNA revealed that 27 out of 29 proteins were in fact N‐myristoylated. Database searches with these 27 cDNA clones revealed that 18 out of 27 proteins are novel N‐myristoylated proteins that have not been reported previously to be N‐myristoylated, indicating that this strategy is useful for the comprehensive identification of human N‐myristoylated proteins from human cDNA resources.     

Recombinant tagging system using ribosomal frameshifting to monitor protein expression

For rapid and accurate quantitation of recombinant proteins during expression and after purification, we introduce a new tagging strategy that expresses both target proteins and limitedly tagged target proteins together in a single cell at a constant ratio by utilizing cis‐elements of programmed ‐1 ribosomal frameshifting (‐1RFS) as an embedded device. ‐1RFS is an alternative reading mechanism that effectively controls protein expression by many viruses. When a target gene is fused to the enhanced green fluorescent protein (EGFP) gene with a ‐1RFS element implanted between them, the unfused target and the target‐GFP fusion proteins are expressed at a fixed ratio. The expression ratio between these two protein products is adjustable simply by changing ‐1RFS signals. This limited‐tagging system would be valuable for the real‐time monitoring of protein expression when optimizing expression condition for a new protein, and in monitoring large‐scale bioprocesses without a large metabolic burden on host cells. Furthermore, this strategy allows for the direct measurement of the quantity of a protein on a chip surface and easy application to proteomewide study of gene products. Biotechnol. Bioeng. 2013; 110: 898–904. © 2012 Wiley Periodicals, Inc.     

Small‐angle X‐ray and neutron scattering demonstrates that cell‐free expression produces properly formed disc‐shaped nanolipoprotein particles

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native‐like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent‐solubilized protein‐lipid mixture. Recently, an alternative method has been developed using direct cell‐free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell‐free expression method are structurally indistinguishable from those produced using detergent removal methodologies. This further supports the utility of single step cell‐free methods for the production of lipid binding proteins. In addition, detailed structural information describing these NLPs can be obtained by fitting a capped core‐shell cylinder type model to all SANS/SAXS data simultaneously.     

Genomewide identification and analysis of heat‐shock proteins 70/110 to reveal their potential functions in Chinese soft‐shelled turtle Pelodiscus sinensis

Heat‐shock proteins 70/110 (Hsp70/110) are vital molecular chaperones and stress proteins whose expression and production are generally induced by extreme temperatures or external stresses. The Hsp70/110 family is largely conserved in diverse animals. Although many reports have studied and elaborated on the characteristics of Hsp70/110 in various species, the systematic identification and analysis of Hsp70/110 are still poor in turtles. In this study, a genomewide search was performed, and 18 candidate PsHSP70/110 family genes were identified in Chinese soft‐shelled turtle, Pelodiscus sinensis. These PsHSP70/110 proteins contained the conserved “heat shock protein 70” domain. Phylogenetic analysis of PsHSP70/110 and their homologs revealed evolutionary conservation of Hsp70/110 across different species. Tissue‐specific expression analysis showed that these PsHSP70/110 genes were differentially expressed in different tissues of P. sinensis. Furthermore, to examine the putative biological functions of PsHSP70/110, the dynamic expression of PsHSP70/110 genes was analyzed in the testis of P. sinensis during seasonal spermatogenesis following germ cell apoptosis. Notably, genes such as PsHSPA1B‐L, PsHSPA2, and PsHSPA8 were significantly upregulated in P. sinensis testes along with a seasonal decrease in apoptosis. Protein interaction prediction revealed that PsHSPA1B‐L, PsHSPA2, and PsHSPA8 may interact with each other and participate in the MAPK signaling pathway. Moreover, immunohistochemical analysis showed that PsHSPA1B‐L, PsHSPA2, and PsHSPA8 protein expression was associated with seasonal temperature variation. The expression profiling and interaction relationships of the PsHSPA1B‐L, PsHSPA2, and PsHSPA8 proteins implied their potential roles in inhibiting the apoptosis of germ cells in P. sinensis. These results provide insights into PsHSP70/110 functions and will serve as a rich resource for further investigation of HSP70/110 family genes in P. sinensis and other turtles.     

SWATH enables precise label‐free quantification on proteome scale

MS‐based proteomics has emerged as a powerful tool in biological studies. The shotgun proteomics strategy, in which proteolytic peptides are analyzed in data‐dependent mode, enables a detection of the most comprehensive proteome (>10 000 proteins from whole‐cell lysate). The quantitative proteomics uses stable isotopes or label‐free method to measure relative protein abundance. The isotope labeling strategies are more precise and accurate compared to label‐free methods, but labeling procedures are complicated and expensive, and the sample number and types are also limited. Sequential window acquisition of all theoretical mass spectra (SWATH) is a recently developed technique, in which data‐independent acquisition is coupled with peptide spectral library match. In principle SWATH method is able to do label‐free quantification in an MRM‐like manner, which has higher quantification accuracy and precision. Previous data have demonstrated that SWATH can be used to quantify less complex systems, such as spiked‐in peptide mixture or protein complex. Our study first time assessed the quantification performance of SWATH method on proteome scale using a complex mouse‐cell lysate sample. In total 3600 proteins got identified and quantified without sample prefractionation. The SWATH method shows outstanding quantification precision, whereas the quantification accuracy becomes less perfect when protein abundances differ greatly. However, this inaccuracy does not prevent discovering biological correlates, because the measured signal intensities had linear relationship to the sample loading amounts; thus the SWATH method can predict precisely the significance of a protein. Our results prove that SWATH can provide precise label‐free quantification on proteome scale.