Engineered Chimera Protein Constructs to Facilitate the Production of Heterologous Transmembrane Proteins in E. coli.

To delve into the structure-function relationship of transmembrane proteins (TMPs), robust protocols are needed to produce them in a pure, stable, and functional state. Among all hosts that express heterologous TMPs, E. coli has the lowest cost and fastest turnover. However, many of the TMPs expressed in E. coli are misfolded. Several strategies have been developed to either direct the foreign TMPs to E. coli's membrane or retain them in a cytosolic soluble form to overcome this deficiency. Here, we summarize protein engineering methods to produce chimera constructs of the desired TMPs fused to either a signal peptide or precursor maltose binding protein (pMBP) to direct the entire construct to the periplasm, therefore depositing the fused TMP in the plasma membrane. We further describe strategies to produce TMPs in soluble form by utilizing N-terminally fused MBP without a signal peptide. Depending on its N- or C-terminus location, a fusion to apolipoprotein AI can either direct the TMP to the membrane or shield the hydrophobic regions of the TMP, maintaining the soluble form. Strategies to produce G-protein-coupled receptors, TMPs of Mycobacterium tuberculosis, HIV-1 Vpu, and other TMPs are discussed. This knowledge could increase the scope of TMPs' expression in E. coli.

Design and Escherichia coli Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD).

Refolding multi-disulfide bonded proteins expressed in E. coli into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and versatility, the E. coli expression of viral envelope proteins, such as the RBD (Receptor-Binding Domain) of the influenza Hemagglutinin protein, could significantly advance research on viral infections. Here, we show that H1N1-PR8-RBD (27 kDa, containing four cysteines forming two disulfide bonds) expressed in E. coli and was purified with nickel affinity chromatography, and reversed-phase HPLC was successfully refolded into its native structure, as assessed with several biophysical and biochemical techniques. Analytical ultracentrifugation indicated that H1N1-PR8-RBD was monomeric with a hydrodynamic radius of 2.5 nm. Thermal denaturation, monitored with DSC and CD at a wavelength of 222 nm, was cooperative with a midpoint temperature around 55 °C, strongly indicating a natively folded protein. In addition, the 15N-HSQC NMR spectrum exhibited several 1H-15N resonances indicative of a beta-sheeted protein. Our results indicate that a significant amount (40 mg/L) of pure and native H1N1-PR8-RBD can be produced using an E. coli expression system with our refolding procedure, offering potential insights into the molecular characterization of influenza virus infection.

Non-Glycosylated SARS-CoV-2 Omicron BA.5 Receptor Binding Domain (RBD) with a Native-like Conformation Induces a Robust Immune Response with Potent Neutralization in a Mouse Model.

The Omicron BA.5 variant of SARS-CoV-2 is known for its high transmissibility and its capacity to evade immunity provided by vaccine protection against the (original) Wuhan strain. In our prior research, we successfully produced the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein in an E. coli expression system. Extensive biophysical characterization indicated that, even without glycosylation, the RBD maintained native-like conformational and biophysical properties. The current study explores the immunogenicity and neutralization capacity of the E. coli-expressed Omicron BA.5 RBD using a mouse model. Administration of three doses of the RBD without any adjuvant elicited high titer antisera of up to 7.3 × 105 and up to 1.6 × 106 after a booster shot. Immunization with RBD notably enhanced the population of CD44+CD62L+ T cells, indicating the generation of T cell memory. The in vitro assays demonstrated the antisera's protective efficacy through significant inhibition of the interaction between SARS-CoV-2 and its human receptor, ACE2, and through potent neutralization of a pseudovirus. These findings underscore the potential of our E. coli-expressed RBD as a viable vaccine candidate against the Omicron variant of SARS-CoV-2.

High-level expression of functional Pfu DNA polymerase recombinant protein by mimicking the enhanced green fluorescence protein gene codon usage.

Pfu DNA polymerase is a vital enzyme in PCR-related experiments. However, it is not easy to achieve high-level expression and high purity through one-step purification. This paper illustrates the method to acquire the full-length open reading frame of Pfu DNA polymerase. Without altering its amino acids, we have modified the codon usage, based on that of the enhanced green fluorescence protein (eGFP), and named it rPfu. The synthesized rPfu gene has been subcloned into the pET28a plasmid and expressed in four Escherichia coli strains without the pLysS plasmid. Three strains have expressed a high level of soluble Pfu DNA polymerase. With the aid of Ni-NTA His•Bind® resin, we could obtain high purity (>95%) soluble recombinant protein. Compared with the commercial, proofreading DNA polymerase, rPfu's bioactivity was 12,987 U/mg; that is, 88,311 U of rPfu could be obtained from 50 mL cultured E. coli. The purified rPfu was able to amplify the length of DNA fragments at least 5.5 kb. The method of increasing soluble protein's yield using the eGFP codon usage may introduce a new possibility to the expression of other soluble recombinant proteins.

Glycoprotein In Vitro N-Glycan Processing Using Enzymes Expressed in E. coli.

Protein N-glycosylation is a common post-translational modification that plays significant roles on the structure, property, and function of glycoproteins. Due to N-glycan heterogeneity of naturally occurring glycoproteins, the functions of specific N-glycans on a particular glycoprotein are not always clear. Glycoprotein in vitro N-glycan engineering using purified recombinant enzymes is an attractive strategy to produce glycoproteins with homogeneous N-glycoforms to elucidate the specific functions of N-glycans and develop better glycoprotein therapeutics. Toward this goal, we have successfully expressed in E. coli glycoside hydrolases and glycosyltransferases from bacterial and human origins and developed a robust enzymatic platform for in vitro processing glycoprotein N-glycans from high-mannose-type to α2-6- or α2-3-disialylated biantennary complex type. The recombinant enzymes are highly efficient in step-wise or one-pot reactions. The platform can find broad applications in N-glycan engineering of therapeutic glycoproteins.

Tick-borne encephalitis nonstructural protein NS1 expressed in E. coli retains immunological properties of the native protein.

There is evidence that flaviviral NS1 glycoprotein plays an important role in the pathology of tick-borne encephalitis (TBE) and NS1-specific antibodies are detected in the blood of patients with TBE. This makes NS1 a good target for the development of therapeutic inhibitors and NS1 could be an important biomarker for the early diagnosis of TBE in vaccinated individuals. Eukaryotic expression systems are mainly used to produce recombinant tick-borne encephalitis virus (TBEV) NS1. The expression of TBEV NS1 proteins in eukaryotic cells was successful, but there were some limitations. Several attempts have also been made to obtain the NS1 protein in Escherichia coli cells; however, they were unsuccessful due to the low solubility of the recombinant protein and improper folding. In this study, using Trx-tag as a fusion partner, soluble Trx-fused TBEV NS1 protein was first produced in the E. coli BL21 strain. In addition, insoluble Trx-fused TBEV NS1 protein was obtained when cultivation conditions were changed to increase the productivity. The insoluble TBEV NS1 obtained from inclusion bodies was solubilized using chaotropic reagents and successfully refolded using dialysis. Both soluble variant and successfully refolded from inclusion bodies variant showed immunological properties similar to the native TBEV NS1 protein and were recognized by specific monoclonal antibodies (mAbs), immune ascetic fluid in ELISA, western blot, and competitive analysis.

Efficient production of a functional G protein-coupled receptor in E. coli for structural studies.

G protein-coupled receptors (GPCRs) are transmembrane signal transducers which regulate many key physiological process. Since their discovery, their analysis has been limited by difficulties in obtaining sufficient amounts of the receptors in high-quality, functional form from heterologous expression hosts. Albeit highly attractive because of its simplicity and the ease of isotope labeling for NMR studies, heterologous expression of functional GPCRs in E. coli has proven particularly challenging due to the absence of the more evolved protein expression and folding machinery of higher eukaryotic hosts. Here we first give an overview on the previous strategies for GPCR E. coli expression and then describe the development of an optimized robust protocol for the E. coli expression and purification of two mutants of the turkey ß1-adrenergic receptor (ß1AR) uniformly or selectively labeled in 15N or 2H,15N. These mutants had been previously optimized for thermal stability using insect cell expression and used successfully in crystallographic and NMR studies. The same sequences were then used for E. coli expression. Optimization of E. coli expression was achieved by a quantitative analysis of losses of receptor material at each step of the solubilization and purification procedure. Final yields are 0.2-0.3 mg receptor per liter culture. Whereas both expressed mutants are well folded and competent for orthosteric ligand binding, the less stable YY-ß1AR mutant also comprises the two native tyrosines Y5.58 and Y7.53, which enable G protein binding. High-quality 1H-15N TROSY spectra were obtained for E. coli-expressed YY-ß1AR in three different functional states (antagonist, agonist, and agonist + G protein-mimicking nanobody-bound), which are identical to spectra obtained of the same forms of the receptor expressed in insect cells. NdeI and AgeI restriction sites introduced into the expression plasmid allow for the easy replacement of the receptor gene by other GPCR genes of interest, and the provided quantitative workflow analysis may guide the respective adaptation of the purification protocol.

High-yield production of active recombinant S. simulans lysostaphin expressed in E. coli in a laboratory bioreactor.

Staphylococcus aureus (S. aureus), which has developed multidrug resistance, leads to many healthcare-associated infections resulting in significant medical and economic losses. Therefore, the development of new efficient strategies to deal with these bacteria has been gaining importance. Lysostaphin is a peptidoglycan hydrolase that has considerable potential as a bacteriocin. However, there have been few reported optimization and scale-up studies of the lysostaphin bioproduction process. Our preliminary results have revealed that the composition of auto-induction media at 30 °C increases the produced lysostaphin around 10-fold in shake flasks. In this study, achieving higher yields for recombinant lysostaphin in E. coli at a laboratory scale has been the aim, through the use of auto-induction media. Optimized medium composition and fermentation parameters were transferred to a laboratory-scale bioreactor. The tested conditions improved protein yields up to 184 mg/L in a 3 L stirred bioreactor and the productivity was improved 2-fold in comparison to previously published reports. Furthermore, this study also showed that lysostaphin is an effective bacteriocin on both commercially available and isolated S. aureus strains. These results will contribute to future larger-scale production of lysostaphin via the proposed fermentation conditions.

Purification and characterization of an FeII- and α-ketoglutarate-dependent xanthine hydroxylase from Aspergillus oryzae.

XanA is an FeII- and α-ketoglutarate-dependent enzyme responsible for the conversion of xanthine to uric acid. It is unique to fungi and it was first described in Aspergillus nidulans. In this work, we present the preliminary characterization of the XanA enzyme from Aspergillus oryzae, a relevant fungus in food production in Japan. The XanA protein (GenBank BAE56701.1) was expressed as a recombinant protein in Escherichia coli BL21 (DE3) Arctic cells. Initial purification assays showed low protein solubility; therefore, the buffer composition was optimized using a fluorescence-based thermal shift assay. The protein was stabilized in solution in the presence of either 600 µM xanthine, 1 M NaCl, 600 µM α-ketoglutarate or 20% glycerol, which increases the melting temperature (Tm) by 2, 4, 5 and 6 °C respectively. The XanA protein was purified by following a three-step purification protocol. The nickel affinity purified protein was subjected to ion-exchange chromatography once the N-terminal 6XHis-tag had been successfully removed, followed by size-exclusion purification. Dynamic light scattering experiments showed that the purified protein was monodisperse and behaved as a monomer in solution. Preliminary activity assays in the presence of xanthine, α-ketoglutarate, and iron suggest that the enzyme is an iron- and α-ketoglutarate-dependent xanthine dioxygenase. Furthermore, the enzyme's optimum activity conditions were determined to be 25 °C, pH of 7.2, HEPES buffer, and 1% of glycerol. In conclusion, we established the conditions to purify the XanA enzyme from A. oryzae in its active form from E. coli bacteria and determined the optimal activity conditions.

Production of bioactive recombinant human myeloid-derived growth factor in Escherichia coli and its mechanism on vascular endothelial cell proliferation.

Myeloid-derived growth factor (MYDGF) is a novel protein secreted by bone marrow cells that features important physiological functions. In recent years, MYDGF has gained considerable interest due to their extensive beneficial effect on cardiac repair and protects cardiomyocytes from cell death. However, its precise molecular mechanisms have not been well elucidated. The purpose of this study was to produce sufficient amount of biologically active recombinant human (rh) MYDGF more economically and effectively by using in vitro molecular cloning techniques to study its clinical application. The prokaryotic expression system of Escherichia coli was established for the preparation of rhMYDGF. Finally, a large amount of high biologically active and purified form of recombinant protein was obtained. Moreover, we investigated the potential mechanism of rhMYDGF-mediated proliferation and survival in human coronary artery endothelial cells (HCAECs). Mechanistically, the results suggested that MAPK/STAT3 and the cyclin D1 signalling pathways are indispensable for rhMYDGF-mediated HCAEC proliferation and survival. Therefore, this study successfully established a preparation protocol for biologically active rhMYDGF and it may be a most economical way to produce high-quality active rhMYDGF for future clinical application.

Expression and purification of amyloid ß-protein, tau, and α-synuclein in Escherichia coli: a review.

Misfolding and accumulation of amyloidogenic proteins into various forms of aggregated intermediates and insoluble amyloid fibrils is associated with more than 50 human diseases. Large amounts of high-quality amyloid proteins are required for better probing of their aggregation and neurotoxicity. Due to their intrinsic hydrophobicity, it is a challenge to obtain amyloid proteins with high yield and purity, and they have attracted the attention of researchers from all over the world. The rapid development of bioengineering technology provides technical support for obtaining large amounts of recombinant amyloidogenic proteins. This review discusses the available expression and purification methods for three amyloid proteins including amyloid ß-protein, tau, and α-synuclein in microbial expression systems, especially Escherichia coli, and discusses the advantages and disadvantages of these methods. Importantly, these protocols can also be referred to for the expression and purification of other hydrophobic proteins.

Development of a recombinant VP2 vaccine for the prevention of novel variant strains of infectious bursal disease virus.

Since 2017, novel variant strains of infectious bursal disease virus (nvIBDV) have been detected in China, while the current vaccines on the market against very virulent IBDV have limited protection against this subtype virus. In this context, a strain of the virus has been isolated, and sequencing alignment and bird regression experiments showed that the virus was IBDV, belonging to the nvIBDV subtype (and named IBDV FJ-1812). Furthermore, the Escherichia coli expression system was used to successfully express soluble nvIBDV rVP2, which is specifically recognized by an anti-IBDV standard serum and anti-nvIBDV positive serum, and could be assembled into 14 - 17 nm virus-like particles. Based on the purified nvIBDV rVP2, we developed an IBDV FJ-1812 VP2 VLP vaccine at a laboratory scale to evaluate protection by this vaccine; in addition, we also prepared an IBDV JZ 3/02 VP2 subunit vaccine targeting very virulent IBDV and evaluated its cross-protection against nvIBDV. Results of bird experiments showed that the nvIBDV rVP2 vaccine could induce high titres of specific antibodies, completely protect the bursa of Fabricius from viral infection, and provide 100% immune protection to SPF and Ross 308 broiler chickens. Furthermore, the IBDV JZ 3/02 VP2 subunit vaccine targeting very virulent IBDV could provide 60% protection for SPF chickens and 80% protection for Ross 308 broiler chickens. This report provides important technical supports for the prevention and control of nvIBDV in the future.

Enhanced Heterologous Production of Glycosyltransferase UGT76G1 by Co-Expression of Endogenous prpD and malK in Escherichia coli and Its Transglycosylation Application in Production of Rebaudioside.

Steviol glycosides (SGs) with zero calories and high-intensity sweetness are the best substitutes of sugar for the human diet. Uridine diphosphate dependent glycosyltransferase (UGT) UGT76G1, as a key enzyme for the biosynthesis of SGs with a low heterologous expression level, hinders its application. In this study, a suitable fusion partner, Smt3, was found to enhance the soluble expression of UGT76G1 by 60%. Additionally, a novel strategy to improve the expression of Smt3-UGT76G1 was performed, which co-expressed endogenous genes prpD and malK in Escherichia coli. Notably, this is the first report of constructing an efficient E. coli expression system by regulating prpD and malK expression, which remarkably improved the expression of Smt3-UGT76G1 by 200% as a consequence. Using the high-expression strain E. coli BL21 (DE3) M/P-3-S32U produced 1.97 g/L of Smt3-UGT76G1 with a yield rate of 61.6 mg/L/h by fed-batch fermentation in a 10 L fermenter. The final yield of rebadioside A (Reb A) and rebadioside M (Reb M) reached 4.8 g/L and 1.8 g/L, respectively, when catalyzed by Smt3-UGT76G1 in the practical UDP-glucose regeneration transformation system in vitro. This study not only carried out low-cost biotransformation of SGs but also provided a novel strategy for improving expression of heterologous proteins in E. coli.

Cloning and characterization of a L-lactate dehydrogenase gene from Ruminococcaceae bacterium CPB6.

Lactate are proved to be attractive electron donor for the production of n-caproic acid (CA) that is a high value-added fuel precursor and chemical feedstock, but little is known about molecular mechanism of lactate transformation. In the present study, the gene for L-lactate dehydrogenase (LDH, EC.1.1.1.27) from a Ruminococcaceae strain CPB6 was cloned and expressed in Escherichia coli BL21 (DE3) with plasmid pET28a. The recombinant LDH exhibited molecular weight of 36-38 kDa in SDS-PAGE. The purified LDH was found to have the maximal oxidation activity of 29.6 U/mg from lactate to pyruvate at pH 6.5, and the maximal reduction activity of 10.4 U/mg from pyruvate to lactate at pH 8.5, respectively. Strikingly, its oxidative activity predominates over reductive activity, leading to a 17-fold increase for the utilization of lactate in E. coli/pET28a-LDH than E. coli/pET28a. The CPB6 LDH gene encodes a 315 amino acid protein sharing 42.19% similarity with Clostridium beijerinckii LDH, and lower similarity with LDHs of other organisms. Significant difference were observed between the CPB6 LDH and C. beijerinckii and C. acetobutylicum LDH in the predicted tertiary structure and active center. Further, X-ray crystal structure analysis need to be performed to verify the specific active center of the CPB6 LDH and its role in the conversion of lactate into CA.

Generation of single-chain Fv antibody fragments against Mu-2-related death-inducing gene in Escherichia coli.

Mu-2-related death-inducing (MuD) gene is involved in apoptosis in tumor cells. Although we have previously produced mouse monoclonal antibodies (MAbs) that specifically recognize human MuD, the application scope of MuD MAbs was restricted due to their mouse origin. Therefore, we attempted the generation of single-chain variable fragment (scFv) against MuD. The heavy- and light-chain variable region genes from two MuD hybridomas were isolated by PCR and joined by DNA encoding a (Gly4Ser1)3 linker. These scFv fragments were cloned into a phagemid vector and expressed as E-tagged fusion proteins in Escherichia coli HB2151. The reactivity of selected Abs was evaluated using ELISA. Selected MuDscFv Abs specifically recognized human MuD, retaining ~ 50% potency of the parent MAbs. MuDscFv-M3H9 recognized the middle region of MuD, while MuDscFv-C22B3 recognized a broad region. Intracellular expression of MuDscFvs-C22B3 protected cells from TRAIL-induced apoptosis. These MuDscFv Abs may help in the study of intracellular signaling pathway centered on MuD and of drug use target and points.

Characterization and improvement of substrate-binding affinity of D-aspartate oxidase of the thermophilic fungus Thermomyces dupontii.

D-Aspartate oxidase (DDO) is a valuable enzyme that can be utilized in the determination of acidic D-amino acids and the optical resolution of a racemic mixture of acidic amino acids, which require its higher stability, higher catalytic activity, and higher substrate-binding affinity. In the present study, we identified DDO gene (TdDDO) of a thermophilic fungus, Thermomyces dupontii, and characterized the recombinant enzyme expressed in Escherichia coli. In addition, we generated a variant that has a higher substrate-binding affinity. The recombinant TdDDO expressed in E. coli exhibited oxidase activity toward acidic D-amino acids and a neutral D-amino acid, D-Gln, with the highest activity toward D-Glu. The Km and kcat values for D-Glu were 2.16 mM and 217 s-1, respectively. The enzyme had an optimum pH and temperature 8.0 and 60 °C, respectively, and was stable between pH 5.0 and 10.0, with a T50 of ca. 51 °C, which was much higher than that in DDOs from other origins. Enzyme stability decreased following a decrease in protein concentration, and externally added FAD could not repress the destabilization. The mutation of Phe248, potentially located in the active site of TdDDO, to Tyr residue, conserved in DDOs and D-amino acid oxidases, markedly increased substrate-binding affinity. The results showed the great potential of TdDDO and the variant for practical applications.

High Expression Achievement of Active and Robust Anti-ß2 microglobulin Nanobodies via E.coli Hosts Selection.

Nanobodies (VHHs) overcome many of the drawbacks of conventional antibodies, and the related technologies represent state-of-the-art and advanced applications in scientific research, pharmaceuticals, and therapies. In terms of productivity and economic cost, the cytoplasmic expression of VHHs in Escherichia coli (E. coli) is a good process for their recombinant production. The cytoplasmic environment of the host is critical to the affinity and stability of the recombinant VHHs in soluble form, yet the effects have not been studied. For this purpose, recombinant anti-ß2 microglobulin VHHs were constructed and expressed in four commercialized E. coli hosts, including BL21 (DE3), Rosetta-gami B (DE3) pLysS, Origami 2 (DE3) and SHuffle T7 Express. The results showed that anti-ß2 microglobulin (ß2MG) VHHs expressed in different hosts exhibited distinctive differences in the affinity and structural characteristics. The VHHs expressed in Rosetta-gami B (DE3) pLysS possessed not only the greatest affinity of (equilibrium dissociation constant) KD = 4.68 × 10-8 M but also the highest yields compared with the VHHs expressed in BL21 (DE3), Origami 2 (DE3) and SHuffle T7 Express. In addition, the VHHs expressed in Rosetta-gami B (DE3) pLysS were more stable than the VHHs expressed in the rest three hosts. Thus far, we have successfully realized the high expression of the active and robust anti-ß2MG VHHs in Rosetta-gami B (DE3) pLysS. The underlying principle of our study is able to guide the expression strategies of nanobodies on the context of industrial large-scale production.

Generation of a fluorescent oncoprotein in soluble form and its delivery into mammalian cells.

BACKGROUND: Protein fusion technology was widely used to improve expression, purification and solubility of the recombinant proteins expressed in E. coli for in vitro/in vivo delivery. METHODS: We developed a method for successful expression of soluble +36GFP-A2-E7 protein in E. coli and its effective delivery into mammalian cells. At first, the plasmid harboring +36GFP-A2-E7 was transformed into E. coli Rosetta competent cells. Then, the recombinant protein fused to histidine tag was expressed and purified using affinity chromatography. Different conditions such as inducer dose, time and temperature of induction, pH and urea concentration were evaluated. Finally, the delivery of the recombinant protein was detected in HEK-293T cells using fluorescent microscopy and flow cytometry. RESULTS: Our data showed that the expressed protein formed inclusion bodies at 37 °C and 3 h post-induction. The soluble protein was generated using 0.5 mM IPTG and growth at 16 °C for 20 h, and purified by low concentrations of urea and 200 mM imidazole. The soluble fraction of +36GFP-A2-E7 protein could significantly represent higher fluorescent property and stronger delivery into mammalian cells compared to the insoluble form. CONCLUSION: Generally, soluble form of fusion protein retained its biological activity and could directly penetrate into the cells without the fusion tags (Tab. 1, Fig. 7, Ref. 23).

Facile preparation of highly active casein kinase 1 using Escherichia coli constitutively expressing lambda phosphatase.

Casein kinase 1 (CK1) is a widely expressed Ser/Thr kinase in eukaryotic organisms that is involved in various cellular processes (e.g., circadian rhythm and apoptosis). Therefore, preparing highly active CK1 and investigating its properties in vitro have important implications for understanding the biological roles of the kinase. However, recombinant CK1 undergoes autoinactivation via autophosphorylation in Escherichia coli cells and thus is undesirably prepared as a phosphorylated and inactivated kinase. To circumvent this problem, we established a protein expression system using E. coli strain BL21(DE3)pλPP in which λ protein phosphatase (λPPase) is constitutively expressed. Using this system, recombinant CK1 isoforms (α, δ and ε) were readily prepared as unphosphorylated forms. Furthermore, we found that CK1s prepared using BL21(DE3)pλPP showed markedly higher activity than those prepared by the conventional BL21(DE3). Finally, we demonstrated that the kinase activity of CK1δ from BL21(DE3)pλPP was higher than that prepared by a conventional method consisting of troublesome steps such as in vitro λPPase treatment. Thus, this simple method using BL21(DE3)pλPP is valuable for preparing highly active CK1s. It may also be applicable to other kinases that are difficult to prepare because of phosphorylation in E. coli cells.

Generation of Nanobodies against SlyD and development of tools to eliminate this bacterial contaminant from recombinant proteins.

The gene for a protein domain, derived from a tumor marker, fused to His tag codons and under control of a T7 promotor was expressed in E. coli strain BL21 (DE3). The recombinant protein was purified from cell lysates through immobilized metal affinity chromatography and size-exclusion chromatography. A contaminating bacterial protein was consistently co-purified, even using stringent washing solutions containing 50 or 100 mM imidazole. Immunization of a dromedary with this contaminated protein preparation, and the subsequent generation and panning of the immune Nanobody library yielded several Nanobodies of which 2/3 were directed against the bacterial contaminant, reflecting the immunodominance of this protein to steer the dromedary immune response. Affinity adsorption of this contaminant using one of our specific Nanobodies followed by mass spectrometry identified the bacterial contaminant as FKBP-type peptidyl-prolyl cis-trans isomerase (SlyD) from E. coli. This SlyD protein contains in its C-terminal region 14 histidines in a stretch of 31 amino acids, which explains its co-purification on Ni-NTA resin. This protein is most likely present to varying extents in all recombinant protein preparations after immobilized metal affinity chromatography. Using our SlyD-specific Nb 5 we generated an immune-complex that could be removed either by immunocapturing or by size exclusion chromatography. Both methods allow us to prepare a recombinant protein sample where the SlyD contaminant was quantitatively eliminated.