Gene cloning,bacterial expression,and purification of calreticulin from Anopheles stephensi (AsCRT)

During the invasion of Plasmodium ookinetes to the mosquito midgut epithelium, several proteins or glycoproteins are involved. Recent study has shown that the calreticulin (CRT) of the midgut from Anopheles albimanus can bind to the protein receptor Pvs25 on surface of Plasmodium vivax ookinetes. Thus, in order to get more insight into the potential roles of Anopheles stephensi calreticulin (AsCRT) in the midgut, we amplified and cloned the full‐length of calreticulin coding sequence from Anopheles stephensi. The AsCRT consists of 1221 bp nucleic acids with one open reading frame (ORF) encoding 406 amino acids and an apparent molecular weight around 46 KDa. Subsequently, the recombinant calreticulin as Glutathione S‐transferase (GST) fusion in pGEX ?6p‐1 expression vector (GST‐AsCRT) was produced in the prokaryotic system under optimum conditions. GST‐AsCRT fusion protein has a molecular weight around 73 KDa. The recombinant protein was detected by Western blotting using a rabbit anti‐GST polyclonal antibody. Here, we report via single protein purification procedure using MagneGST beads, 25 mg of the recombinant protein was obtained per liter of bacterial culture. This is the first report describing the heterologous expression of Anopheles stephensi calreticulin in the prokaryotic system. The production of this recombinant protein will now allow us to further investigate AsCRT molecular protein analyses, characterization of physiochemical properties, as well as interaction between calreticulin and plasmodium protein surface.     

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.     

Heterologous expression and purification of active L‐asparaginase I of Saccharomyces cerevisiae in Escherichia coli host

l ‐asparaginase (ASNase) is a biopharmaceutical widely used to treat child leukemia. However, it presents some side effects, and in order to provide an alternative biopharmaceutical, in this work, the genes encoding ASNase from Saccharomyces cerevisiae (Sc_ASNaseI and Sc_ASNaseII) were cloned in the prokaryotic expression system Escherichia coli. In the 93 different expression conditions tested, the Sc_ASNaseII protein was always obtained as an insoluble and inactive form. However, the Sc_ASNaseI (His)₆‐tagged recombinant protein was produced in large amounts in the soluble fraction of the protein extract. Affinity chromatography was performed on a Fast Protein Liquid Chromatography (FPLC) system using Ni²⁺‐charged, HiTrap Immobilized Metal ion Affinity Chromatography (IMAC) FF in order to purify active Sc_ASNaseI recombinant protein. The results suggest that the strategy for the expression and purification of this potential new biopharmaceutical protein with lower side effects was efficient since high amounts of soluble Sc_ASNaseI with high specific activity (110.1 ± 0.3 IU mg^?1) were obtained. In addition, the use of FPLC‐IMAC proved to be an efficient tool in the purification of this enzyme, since a good recovery (40.50 ± 0.01%) was achieved with a purification factor of 17‐fold. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:416–424, 2017     

Cloning, expression and purification of extracellular serine protease Esp, a biofilm-degrading enzyme, from Staphylococcus epidermidis

Aims: Staphylococcus epidermidis Esp, an extracellular serine protease, inhibits Staphylococcus aureus biofilm formation and nasal colonization. To further expand the biotechnological applications of Esp, we developed a highly efficient and economic method for the purification of recombinant Esp based on a Brevibacillus choshinensis expression–secretion system. Methods and Results: The esp gene was fused with the N‐terminal Sec‐dependent signal sequence of the B. choshinensis cell wall protein and a C‐terminal hexa‐histidine‐tag gene. The recombinant Esp was expressed and secreted into the optimized medium as an immature form and subsequently activated by thermolysin. The mature Esp was easily purified by a single purification step using nickel affinity chromatography and showed proteolytic activity as well as Staph. aureus biofilm destruction activity. Conclusions: The purification yield of the developed extracellular production system was 5 mg recombinant mature Esp per 20‐ml culture, which was much higher than that of an intracellular production system in Escherichia coli (3 mg recombinant Esp per 1‐l culture). Significance and Impact of the Study: Our findings will be a powerful tool for the production and purification of recombinant Esp and also applicable to a large variety of recombinant proteins used for basic researches and biotechnological applications.     

Process integration for recovery of recombinant collagen type I α1 from corn seed

Because of safety concerns and product consistency issues with the use of animal‐derived collagen, several recombinant protein expression hosts have been considered for recombinant collagen corn seed. Full length, triple‐helical, recombinant collagen (rCIα1) is expressed as a fusion with a foldon domain, which must later be removed. Here we have examined integration of purification and foldon removal by comparing advantages of removal before or after purification, using salt precipitation as the main purification step. Because expression levels in available maize lines are low, Pichia‐produced recombinant collagens, both with and without foldon, were added to corn seed germ at the extraction step. Salt precipitation of an acidic corn seed extract yielded 100% of the collagen without foldon at >70% purity without the pepsin pretreatment. With pepsin pretreatment, yield was 94.0% with purity of 76.5%. Analysis of the protein molecular weight distribution of the pre‐ and post‐treatment extracts showed that the corn proteins are largely resistant to pepsin proteolysis, explaining why little benefit was obtained by pepsin treatment. In the absence of pepsin treatment, the recovery of rCIα1 with foldon was still above 90% but the purity was only 44%. This still represented at about 13‐fold purification with a 2.7‐fold volume reduction which would reduce the pepsin requirement for post‐recovery foldon cleavage. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:98–107, 2016     

Optimized E. coli expression strain LOBSTR eliminates common contaminants from His‐tag purification

His‐tag affinity purification is one of the most commonly used methods to purify recombinant proteins expressed in E. coli. One drawback of using the His‐tag is the co‐purification of contaminating histidine‐rich E. coli proteins. We engineered a new E. coli expression strain, LOBSTR (lo w b ackground str ain), which eliminates the most abundant contaminants. LOBSTR is derived from the E. coli BL21(DE3) strain and carries genomically modified copies of arnA and slyD, whose protein products exhibit reduced affinities to Ni and Co resins, resulting in a much higher purity of the target protein. The use of LOBSTR enables the pursuit of challenging low‐expressing protein targets by reducing background contamination with no additional purification steps, materials, or costs, and thus pushes the limits of standard His‐tag purifications. Proteins 2013; 81:1857–1861. © 2013 Wiley Periodicals, Inc.     

Use of the Recombinant 38-kDa Antigen of Mycobacterium tuberculosis as an Immunogen for Specific Antisera Production

The Mycobacterium tuberculosis 38-kDa protein antigen is one of the secreted immunodominant antigens showing high immunogenicity at B-cell and T-cell levels. Although monoclonal antibodies to this antigen have been produced, specific polyclonal antisera is required for standardization of specific immunodiagnostic assays. This protein has been overexpressed and purified from recombinant Escherichia coli using an inducible vector system. During each stage of expression and purification, the recombinant protein was used to immunize mice and rabbits by several methods: 1) as overexpressed protein present as inclusion bodies in recombinant E. coli; 2) embedded in a polyacrylamide gel; 3) fixed to a solid-phase nitrocellulose membrane and 4) emulsified with an adjuvant. All strategies yielded specific antisera as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses. The results obtained, both quantitative (ELISA) and qualitative (immunoblot) demonstrate that the purified recombinant antigen retains its antigenicity and immunogenicity throughout the various steps in the process of expression and purification and serves as a potent antigen for production of specific antisera to be used in immunoassays.     

Enhanced expression of cysteine‐rich antimicrobial peptide snakin‐1 in Escherichia coli using an aggregation‐prone protein coexpression system

Snakin‐1 (SN‐1) is a cysteine‐rich plant antimicrobial peptide and the first purified member of the snakin family. SN‐1 shows potent activity against a wide range of microorganisms, and thus has great biotechnological potential as an antimicrobial agent. Here, we produced recombinant SN‐1 in Escherichia coli by a previously developed coexpression method using an aggregation‐prone partner protein. Our goal was to increase the productivity of SN‐1 via the enhanced formation of insoluble inclusion bodies in E. coli cells. The yield of SN‐1 by the coexpression method was better than that by direct expression in E. coli cells. After refolding and purification, we obtained several milligrams of functionally active SN‐1, the identity of which was verified by MALDI‐TOF MS and NMR studies. The purified recombinant SN‐1 showed effective antimicrobial activity against test organisms. Our studies indicate that the coexpression method using an aggregation‐prone partner protein can serve as a suitable expression system for the efficient production of functionally active SN‐1. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1520–1528, 2017     

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.     

Enhanced expression,detection and purification of recombinant proteins using RNA stem loop and tandem fusion tags

The creation of a double His-tag fusion that forms a RNA stem loop in the mRNA encoding the N-terminus of the target protein is a novel approach for the enhancement of expression, purification, and detection of a recombinant protein. Compared to a single His-tag fusion, a tandem His-tag fusion RNA stem loop, located downstream of the constitutive groE and Ch promoters, enhanced heterologous gene expression in Brucella, Salmonella, and Escherichia. We demonstrated one-step detection and purification of recombinant green fluorescence protein (GFP) directly from Brucella spp. without using Escherichia coli as an expression host. The amount of purified GFP using the tandem His-tag RNA stem loop increased more than threefold; moreover, the sensitivity of detection increased more than fourfold in comparison to the single His-tag fusion form. This method has the potential to significantly improve heterologous gene expression and high-throughput protein synthesis and purification.     

LPS removal from an E. coli fermentation broth using aqueous two‐phase micellar system

In biotechnology, endotoxin (LPS) removal from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of bacterial expression systems widely used to manufacture therapeutic proteins. The viability of large‐scale industrial production of recombinant biomolecules of pharmaceutical interest significantly depends on the separation and purification techniques used. The aim of this work was to evaluate the use of aqueous two‐phase micellar system (ATPMS) for endotoxin removal from preparations containing recombinant proteins of pharmaceutical interest, such as green fluorescent protein (GFPuv). Partition assays were carried out initially using pure LPS, and afterwards in the presence of E. coli cell lysate. The ATPMS technology proved to be effective in GFPuv recovery, preferentially into the micelle‐poor phase (K_GFPuv < 1.00), and LPS removal into the micelle‐rich phase (%REM_LPS > 98.00%). Therefore, this system can be exploited as the first step for purification in biotechnology processes for removal of higher LPS concentrations. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

High‐level production of soluble pyrroloquinoline quinone‐dependent glucose dehydrogenase in Escherichia coli

Pyrroloquinoline quinone‐dependent glucose dehydrogenase (EC1.1.5.2, PQQGDH) has attracted progressive attention due to its application in glucose detection in clinic diagnosis and industrial bioprocess controls. To satisfy its increasing demand, improvement of PQQGDH production derived from Acinetobacter calcoaceticus L.M.D. 79.41 in recombinant Escherichia coli is necessary and is therefore the focus of the current study. Different carbon sources as well as induction conditions were investigated for overexpression of soluble PQQGDH. The results indicate that the target protein was optimally produced with 20 g/L glucose as the substrate. Moreover, the highest expression level (1530 kU/L) was achieved by a novel two‐temperature cultivation strategy in the 10‐L fermentor. This presents a sixfold improvement over previously reported values. After Ni‐NTA affinity chromatography purification, high‐purity enzyme with the specific activity of 5811 U/mg was obtained with a purification yield of 55%. The purified recombinant PQQGDH showed thermal stability and substrate specificity as the native enzyme. In summary, this work provides an alternative production process to overexpress PQQGDH and shows high applicability for large‐scale production of this important glucose dehydrogenase.     

High‐yield recombinant expression of the chicken antimicrobial peptide fowlicidin‐2 in Escherichia coli

The antimicrobial peptide fowlicidin‐2 identified in chicken is a member of the cathelicidins family. The mature fowlicidin‐2 possesses high antibacterial efficacy and lipopolysaccharide (LPS) neutralizing activity, and also represents an excellent candidate as an antimicrobial agent. In the present study, the recombinant fowlicidin‐2 was successfully produced by Escherichia coli (E. coli) recombinant expression system. The gene encoding fowlicidin‐2 with the codon preference of E. coli was designed through codon optimization and synthesized in vitro. The gene was then ligated into the plasmid pET‐32a(+), which features fusion protein thioredoxin at the N‐terminal. The recombinant plasmid was transformed into E. coli BL21(DE3) and cultured in Luria‐Bertani (LB) medium. After isopropyl‐β‐D‐thiogalactopyranoside (IPTG) induction, the fowlicidin‐2 fusion protein was successfully expressed as inclusion bodies. The inclusion bodies were dissolved and successfully released the peptide in 70% formic acid solution containing cyanogen bromide (CNBr) in a single step. After purification by reverse‐phase high‐performance liquid chromatography (RP‐HPLC), ～6.0 mg of fowlicidin‐2 with purity more than 97% was obtained from 1 litre of bacteria culture. The recombinant peptide exhibited high antibacterial activity against the Gram‐positive and Gram‐negative bacteria, and even drug‐resistant strains. This system could be used to rapidly and efficiently produce milligram quantities of a battery of recombinant antimicrobial peptides as well as for large‐scale production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:369–374, 2015     

Natural and recombinant fungal laccases for paper pulp bleaching

Three laccases, a natural form and two recombinant forms obtained from two different expression hosts, were characterized and compared for paper pulp bleaching. Laccase from Pycnoporus cinnabarinus, a well known lignolytic fungus, was selected as a reference for this study. The corresponding recombinant laccases were produced in Aspergillus oryzae and A. niger hosts using the lacI gene from P. cinnabarinus to develop a production process without using the expensive laccase inducers required by the native source. In flasks, production of recombinant enzymes by Aspergilli strains gave yields close to 80 mg l^–1. Each protein was purified to homogeneity and characterized, demonstrating that the three hosts produced proteins with similar physico-chemical properties, including electron paramagnetic resonance spectra and N-terminal sequences. However, the recombinant laccases have higher Michaelian (K _m) constants, suggesting a decrease in substrate/enzyme affinity in comparison with the natural enzyme. Moreover, the natural laccase exhibited a higher redox potential (around 810 mV), compared with A. niger (760 mV) and A. oryzae (735 mV). Treatment of wheat straw Kraft pulp using laccases expressed in P. cinnabarinus or A. niger with 1-hydroxybenzotriazole as redox mediator achieved a delignification close to 75%, whereas the recombinant laccase from A. oryzae was not able to delignify pulp. These results were confirmed by thioacidolysis. Kinetic and redox potential data and pulp bleaching results were consistent, suggesting that the three enzymes are different and each fungal strain introduces differences during protein processing (folding and/or glycosylation).     

Transgenic tobacco plants as production platform for biologically active human interleukin 2 and its fusion with proteinase inhibitors

Transgenic plants offer a low‐cost approach for the production of pharmaceutically important and commercially valuable recombinant proteins. Our studies were focused on the plant‐based production of human interleukin 2 (hIL‐2) and its fusion with proteinase inhibitors, either SPI2 from Galleria mellonella or CMTI from Cucurbita maxima. Finally, five plant expression cassettes were obtained. Three of them contained the single cDNA encoding CMTI I, SPI2 and hIL‐2, respectively, while two of them contained the translational fusion, SPI2::hIL‐2 and CMTI::hIL‐2. In all cases, the transgenes were controlled by the RbcS1 promoter and terminator and the recombinant proteins were targeted to the endoplasmic reticulum. After tobacco transformation, five groups of transgenic plants were obtained and analysed. The level of recombinant proteins was estimated either by Western blot or by ELISA. The biological activity of plant‐produced hIL‐2 alone or in a fusion with SPI2 or CMTI was confirmed using the mammalian cells proliferation assay. The activities of proteinase inhibitors were confirmed in proteolysis assay using azocoll as a substrate. The usefulness of using proteinase inhibitor CMTI I in a fusion with hIL‐2 as a protective agent against trypsin digestion was demonstrated.     

Production of an anti‐Candida peptide via fed batch and ion exchange chromatography

Interest in peptides as diagnostic and therapeutic materials require their manufacture via either a recombinant or synthetic route. This study examined the former, where a recombinant fusion consisting of an antifungal peptide was expressed and isolated from Escherichia coli. Fed batch fermentation with E. coli harboring an arabinose‐inducible plasmid produced the 12 residue anti‐Candida peptide fused to the N‐terminal of Green Fluorescent Protein (GFP_UV). The purification of the fusion protein, using ion‐exchange chromatography, was monitored by using the intrinsic fluorescence of GFP_UV. The recombinant antifungal peptide was successfully released by cyanogen bromide‐induced cleavage of the fusion protein. The recombinant peptide showed the expected antifungal activity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:865–871, 2016     

Rational design of a fusion partner for membrane protein expression in E. coli

We have designed a novel protein fusion partner (P8CBD) to utilize the co‐translational SRP pathway in order to target heterologous proteins to the E. coli inner membrane. SRP‐dependence was demonstrated by analyzing the membrane translocation of P8CBD‐PhoA fusion proteins in wt and SRP‐ffh77 mutant cells. We also demonstrate that the P8CBD N‐terminal fusion partner promotes over‐expression of a Thermotoga maritima polytopic membrane protein by replacement of the native signal anchor sequence. Furthermore, the yeast mitochondrial inner membrane protein Oxa1p was expressed as a P8CBD fusion and shown to function within the E. coli inner membrane. In this example, the mitochondrial targeting peptide was replaced by P8CBD. Several practical features were incorporated into the P8CBD expression system to aid in protein detection, purification, and optional in vitro processing by enterokinase. The basis of membrane protein over‐expression toxicity is discussed and solutions to this problem are presented. We anticipate that this optimized expression system will aid in the isolation and study of various recombinant forms of membrane‐associated protein.     

Bacterial Expression and Characterization of Functional Recombinant Triosephosphate Isomerase from Schistosoma japonicum

The dimeric enzyme triosephosphate isomerase (TPI) converts glyceraldehyde-3-phosphate to dehydroxyacetone phosphate, a key reaction in glycolysis. Previous studies of the native enzyme in the human bloodflukes belonging to the genus Schistosoma have indicated that TPI is a promising anti-schistosome vaccine antigen. However, a recombinant form of the enzyme is required as an alternative to the impractical option of using biochemically purified TPI obtained from worm tissue for large-scale vaccine use. We previously cloned and sequenced a full-length cDNA encoding the TPI of the Asian (Chinese strain) schistosome Schistosoma japonicum (SjcTPI). We now report very high level bacterial expression of this cDNA and the subsequent purification of the recombinant protein to >98% homogeneity under nondenaturing conditions. The recombinant SjcTPI (re-SjcTPI) was shown to be enzymatically active with a specific activity of 7687 units/mg protein, an activity higher than that of commercially obtained porcine TPI tested concurrently under the same assay conditions. The K_m value for the re-SjcTPI using glyceraldehyde-3-phosphate as substrate was 406.7 μM, which is similar to the K_m values reported for the yeast enzyme and various mammalian TPIs. With the availability of substantial amounts of enzymatically active and readily purified re-SjcTPI made in bacteria we can now test whether the recombinant protein can induce a similar level of protection in vaccination/challenge experiments as the native, biochemically purified enzyme.     

Automation aided optimization of cloning,expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies

The process of obtaining a well‐expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His‐tagged Gateway vector, followed by their small‐scale expression optimization in vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large‐scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.