High quality protein microarray using in situ protein purification

Background  

In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC). This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays.     

Dissection of an old protein reveals a novel application: domain D of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Protein A (sSpAD) as a secretion - tag

Background  

Escherichia coli as a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities. The periplasmic space is often favored for the production of complex proteins due to enhanced disulfide bond formation, increased target product stability and simplified downstream processing. To direct proteins to the periplasmic space rather small proteinaceus tags that can be used for affinity purification would be advantageous.     

A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production

Background  

In the past few years, both automated and manual high-throughput protein expression and purification has become an accessible means to rapidly screen and produce soluble proteins for structural and functional studies. However, many of the commercial vectors encoding different solubility tags require different cloning and purification steps for each vector, considerably slowing down expression screening. We have developed a set of E. coli expression vectors with different solubility tags that allow for parallel cloning from a single PCR product and can be purified using the same protocol.     

Optimization of elastin‐like polypeptide fusions for expression and purification of recombinant proteins in plants

The demand for recombinant proteins for medical and industrial use is expanding rapidly and plants are now recognized as an efficient, inexpensive means of production. Although the accumulation of recombinant proteins in transgenic plants can be low, we have previously demonstrated that fusions with an elastin‐like polypeptide (ELP) tag can significantly enhance the production yield of a range of different recombinant proteins in plant leaves. ELPs are biopolymers with a repeating pentapeptide sequence (VGVPG)_n that are valuable for bioseparation, acting as thermally responsive tags for the non‐chromatographic purification of recombinant proteins. To determine the optimal ELP size for the accumulation of recombinant proteins and their subsequent purification, various ELP tags were fused to green fluorescent protein, interleukin‐10, erythropoietin and a single chain antibody fragment and then transiently expressed in tobacco leaves. Our results indicated that ELP tags with 30 pentapeptide repeats provided the best compromise between the positive effects of small ELP tags (n = 5–40) on recombinant protein accumulation and the beneficial effects of larger ELP tags (n = 80–160) on recombinant protein recovery during inverse transition cycling (ITC) purification. In addition, the C‐terminal orientation of ELP fusion tags produced higher levels of target proteins, relative to N‐terminal ELP fusions. Importantly, the ELP tags had no adverse effect on the receptor binding affinity of erythropoietin, demonstrating the inert nature of these tags. The use of ELP fusion tags provides an approach for enhancing the production of recombinant proteins in plants, while simultaneously assisting in their purification. Biotechnol. Bioeng. 2009;103: 562–573. © 2009 Wiley Periodicals, Inc.     

Automated production of recombinant human proteins as resource for proteome research

Background  

An arbitrary set of 96 human proteins was selected and tested to set-up a fully automated protein production strategy, covering all steps from DNA preparation to protein purification and analysis. The target proteins are encoded by functionally uncharacterized open reading frames (ORF) identified by the German cDNA consortium. Fusion proteins were produced in E. coli with four different fusion tags and tested in five different purification strategies depending on the respective fusion tag. The automated strategy relies on standard liquid handling and clone picking equipment.     

Target selection of soluble protein complexes for structural proteomics studies

Background  

Protein expression in E. coli is the most commonly used system to produce protein for structural studies, because it is fast and inexpensive and can produce large quantity of proteins. However, when proteins from other species such as mammalian are produced in this system, problems of protein expression and solubility arise [1]. Structural genomics project are currently investigating proteomics pipelines that would produce sufficient quantities of recombinant proteins for structural studies of protein complexes. To investigate how the E. coli protein expression system could be used for this purpose, we purified apoptotic binary protein complexes formed between members of the Caspase Associated Recruitment Domain (CARD) family.     

N‐terminal processing of affinity‐tagged recombinant proteins purified by IMAC procedures

The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S‐transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine‐containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7‐triazacyclononane (tacn). The use of this tag‐tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli‐expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP‐1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI‐TOF MS analysis of the cleaved products from the DAP‐1 digestion of the recombinant N‐terminally tagged proteins confirmed the complete removal of the tag within 4‐12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn‐based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli‐expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. Copyright © 2015 John Wiley & Sons, Ltd.     

Challenges and opportunities in the purification of recombinant tagged proteins

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs “tag-ligand” combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established “tag-ligand” systems available for fusion protein purification and also explores current unconventional strategies under development.     

Expression of the affinity tags,glutathione-<Emphasis Type="Italic">S</Emphasis>-transferase and maltose-binding protein,in tobacco chloroplasts

Chloroplast transformation offers an exciting platform for the safe, inexpensive and large-scale production of recombinant proteins in plants. An important advantage for the isolation of proteins produced in the chloroplast would be the use of affinity tags for rapid purification by affinity chromatography. To date, only His-tags have been used. In this study, we have tested the feasibility of expressing two additional affinity tags: glutathione-S-transferase (GST) and a His-tagged derivative of the maltose-binding protein (His₆-MBP). By using the chloroplast 16S rRNA promoter and 5′ untranslated region of phage T7 gene 10, GST and His₆-MBP were expressed in homoplastomic tobacco plants at approximately 7% and 37% of total soluble protein, respectively. GST could be purified by one-step-affinity purification using a glutathione column. Much better recoveries were obtained for His₆-MBP by using a twin-affinity purification procedure involving first immobilised nickel followed by binding to amylose. Interestingly, expression of GST led to cytoplasmic male sterility. Overall, our work expands the tools available for purifying recombinant proteins from the chloroplast.     

A fed‐batch based cultivation mode in Escherichia coli results in improved specific activity of a novel chimeric‐truncated form of tissue plasminogen activator

Aims

A novel chimeric‐truncated form of tissue‐type plasminogen activator (t‐PA) with improved fibrin affinity and resistance to PAI was successfully produced in CHO expression system during our previous studies. Considering advantages of prokaryotic expression systems, the aim in this study was to produce the novel protein in Escherichia coli (BL21) strain and compare the protein potency in batch and fed‐batch processes.

Methods and Results

The expression cassette for the novel t‐PA was prepared in pET‐28a(+). The E. coli expression procedure was compared in traditional batch and newly developed fed batch, EnBase^® Flo system. The protein was purified in soluble format, and potency results were identified using Chromolize t‐PA Assay Kit. The fed‐batch fermentation mode, coupled with a Ni‐NTA affinity purification procedure under native condition, resulted in higher amounts of soluble protein, and about a 30% of improvement in the specific activity of the resulted recombinant protein (46·66 IU mg^?1) compared to traditional batch mode (35·8 IU mg^?1).

Conclusions

Considering the undeniable advantages of expression in the prokaryotic expression systems such as E. coli for recombinant protein production, applying alternative methods of cultivation is a promising approach. In this study, fed‐batch cultivation methods showed the potential to replace miss‐folded formats of protein with proper folded, soluble form with improved potency.

Significance and Impact of the Study

Escherichia coli expression of recombinant proteins still counts for nearly 40% of marketed biopharmaceuticals. The major drawback of this system is the lack of appropriate post‐translational modifications, which may cause potency loss/decline. Therefore, applying alternative methods of cultivation as investigated here is a promising approach to overcome potency decrease problem in this protein production system.     

A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters

Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a correctly folded, functional state and can be purified in amounts suitable for structural investigations.     

High-throughput purification of recombinant proteins using self-cleaving intein tags

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format.     

Purification of recombinant protein by cold‐coacervation of fusion constructs incorporating resilin‐inspired polypeptides

Polypeptides containing between 4 and 32 repeats of a resilin‐inspired sequence AQTPSSYGAP, derived from the mosquito Anopheles gambiae, have been used as tags on recombinant fusion proteins. These repeating polypeptides were inspired by the repeating structures that are found in resilins and sequence‐related proteins from various insects. Unexpectedly, an aqueous solution of a recombinant resilin protein displays an upper critical solution temperature (cold‐coacervation) when held on ice, leading to a separation into a protein rich phase, typically exceeding 200 mg/mL, and a protein‐poor phase. We show that purification of recombinant proteins by cold‐coacervation can be performed when engineered as a fusion partner to a resilin‐inspired repeat sequence. In this study, we demonstrate the process by the recombinant expression and purification of enhanced Green fluorescent protein (EGFP) in E. coli. This facile purification system can produce high purity, concentrated protein solutions without the need for affinity chromatography or other time‐consuming or expensive purification steps, and that it can be used with other bulk purification steps such as low concentration ammonium sulfate precipitation. Protein purification by cold‐coacervation also minimizes the exposure of the target protein to enhanced proteolysis at higher temperature. Biotechnol. Bioeng. 2012; 109: 2947–2954. © 2012 Wiley Periodicals, Inc.     

Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine

Fusion of peptide‐based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram‐range amounts of proteins. IMAC‐Ni(II) columns have become the natural partners of 6xHis‐tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His‐tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur‐containing molecules. In this work, we evaluated two different cysteine‐ and histidine‐containing six amino acid tags linked to the N‐terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine‐containing tagged GFPs were able to bind to IMAC‐Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC‐Ni(II) system reaches less than 20% recovery of the cysteine‐containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC‐Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.     

Binary polypeptide system for permanent and oriented protein immobilization

Background  

Many techniques in molecular biology, clinical diagnostics and biotechnology rely on binary affinity tags. The existing tags are based on either small molecules (e.g., biotin/streptavidin or glutathione/GST) or peptide tags (FLAG, Myc, HA, Strep-tag and His-tag). Among these, the biotin-streptavidin system is most popular due to the nearly irreversible interaction of biotin with the tetrameric protein, streptavidin. The major drawback of the stable biotin-streptavidin system, however, is that neither of the two tags can be added to a protein of interest via recombinant means (except for the Strep-tag case) leading to the requirement for chemical coupling.     

Expression and purification of ELP-intein-tagged target proteins in high cell density <Emphasis Type="Italic">E. coli</Emphasis> fermentation

Background  

Elastin-like polypeptides (ELPs) are useful tools that can be used to non-chromatographically purify proteins. When paired with self-cleaving inteins, they can be used as economical self-cleaving purification tags. However, ELPs and ELP-tagged target proteins have been traditionally expressed using highly enriched media in shake flask cultures, which are generally not amenable to scale-up.     

Removal of cleavage slow points from affinity tags used in the IMAC purification of recombinant proteins

The complete enzymatic removal of affinity tags from tagged recombinant proteins is often required but can be challenging when slow points for cleavage exist. This study documents a general approach to remove N‐terminal tags from recombinant proteins specifically designed to be efficiently captured by IMAC resins. In particular, site‐directed mutagenesis procedures have been used to modify the amino acid sequence of metal binding tags useful in IMAC purifications of recombinant proteins with the objective to increase cleavage efficiency with the exopeptidase, dipeptidyl aminopeptidase 1. These tags were specifically developed for application with borderline metal ions, such as Ni²⁺ or Cu²⁺ ions, chelated to the immobilized ligands, 1,4,7‐triazacyclononane (tacn) and its analogs. Due to the ability to control cleavage site structure and accessibility via site directed mutagenesis methods, these procedures offer considerable scope to obtain recombinant proteins with authentic native N‐termini, thus avoiding any impact on structural stability, humoral and cellular immune responses, or other biological functions. Collectively, these IMAC‐based methods provide a practical alternative to other procedures for the purification of recombinant proteins with tag removal. Overall, this approach is essentially operating as an integrated down‐stream purification capability.     

Multiple affinity purification of a baculovirus-derived recombinant prion protein with in vitro ability to convert to its pathogenic form

We previously showed that baculovirus-derived recombinant prion protein (Bac-PrP) can be converted to the misfolded infectious form (PrP^Sc) by protein misfolding cyclic amplification, an in vitro conversion technique. Bac-PrP, with post-translational modifications, would be useful for various applications such as using PrP as an immunogen for generating anti-PrP antibody, developing anti-prion drugs or diagnostic assays using in vitro conversion systems, and establishing an in vitro prion propagation model. For this purpose, highly purified Bac-PrP with in vitro conversion activity is necessary for use as a PrP^C source, to minimize contamination. Furthermore, an exogenous affinity tag-free form is desirable to avoid potential steric interference by the affinity tags during the conversion process. In this study, we established purification methods for the untagged Bac-PrP under native conditions by combining exogenous double-affinity tags, namely, a polyhistidine-tag and a profinity eXact tag, with an octarepeat sequence of the N-terminal region of PrP, which has metal ion-binding affinity. The untagged Bac-PrP with near-homogeneity was obtained by three-step affinity purification, and it was shown that the final, purified Bac-PrP could convert to its pathogenic form. The presented purification procedure could be applied not only to PrP but also to other eukaryotic, recombinant proteins that require high purity and intact physiological activity.