Guidelines to reach high-quality purified recombinant proteins

The final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the successful downstream application of a recombinant protein depends on its quality. Besides production, which is conditioned by the host, the quality of a recombinant protein product relies mainly on the purification procedure. Thus, the purification strategy must be carefully designed from the molecular level. On the other hand, the quality control of a protein sample must be performed to ensure its purity, homogeneity, and structural conformity, in order to validate the recombinant production and purification process. Therefore, this review aims at providing succinct information on the rational purification design of recombinant proteins produced in Escherichia coli, specifically the tagging purification, as well as on accessible tools for evaluating and optimizing protein quality. The classical techniques for structural protein characterization - denaturing protein gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC), dynamic light scattering (DLS), and circular dichroism (CD) - are revisited with focus on the protein, and their main advantages and disadvantages. Furthermore, methods for determining protein concentration and protein storage are also presented. The guidelines compiled herein will aid preparing pure, soluble and homogeneous functional recombinant proteins from the very beginning of the molecular cloning design.This study was funded by the Fundação para a Ciência e a Tecnologia (FCT), Portugal, under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01- 0145-FEDER-006684) and the Post-Doctoral grant SFRH/BPD/ 110640/2015, and by the BioTecNorte operation (NORTE-01-0145- FEDER-000004) supported by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Produção de β-galactosidase por levedura recombinante : desenvolvimento de um sistema de produção estável

A tecnologia do DNA recombinante é uma ferramenta muito utilizada na produção de roteínas em sistemas heterólogos, proteínas essas de elevado valor acrescentado que são, por natureza, produzidas em baixas quantidades. Com este trabalho pretendeu-se construir estirpes floculantes de Saccharomyces cerevisiae que apresentassem uma produção estável de β-galactosidase de Aspergillus niger por integração do gene que codifica para a expressão desta enzima (lacA) nas sequências δ do genoma desta levedura. Foram utilizados dois sistemas de integração distintos. Um deles utiliza como marca de selecção dominante a resistência ao antibiótico G418 e permite a integração de cópias tandem enquanto que o outro sistema utiliza a marca URA3 e a integração é efectuada em diferentes sítios, com uma única cópia em cada sítio. O gene lacA, ladeado pelo promotor e terminador ADH1, foi clonado em ambos os sistemas que foram seguidamente utilizados para transformar as estirpes S. cerevisiae NCYC869-wt (MatαFlo1) e S. cerevisiae NCYC869-A3 (MatαFlo1ura3), respectivamente. Para o primeiro sistema foram testadas concentrações de antibiótico de 0,2 a 1,5 g/l enquanto que para o segundo sistema foram efectuadas 3 rondas de transformação usando sempre a mesma marca de selecção uma vez que o gene URA3 vai sendo eliminado por recombinação das sequências repetidas que o flanqueiam (hisG). Os transformantes foram seleccionados para estudos posteriores pela actividade de β-galactosidase devido à presença de X-gal nas placas selectivas, ou seja, foram seleccionados pela intensidade de cor azul. Ambos os sistemas de transformação permitiram obter transformantes de S. cerevisiae utilizadores de lactose. Independentemente da marca de selecção utilizada, foram observados diferentes níveis de floculação e diferentes níveis de produção de β-galactosidase. Globalmente, os níveis de produção de β-galactosidase foram mais elevados para o primeiro sistema. Os transformantes que susceptibilizaram maior interesse foram caracterizados geneticamente por hibridação Southern. Para o primeiro sistema, foram observados, no máximo, dois sítios de integração usados em simultâneo com cópias tandem e 8 cópias integradas no total, enquanto que para o segundo, após as 3 rondas de transformação e perda do gene URA3, observou-se apenas dois sítios de integração usados em simultâneo correspondentes a 2 cópias integradas. O melhor transformante seleccionado, pertencente ao primeiro sistema (1,5 g/l de G418), apresenta actividade de β-galactosidase em glucose comparável ao sistema de referência, baseado em plasmídeo epissomal (S. cerevisiae NCYC869-A3/pVK1.1), e apresenta estabilidade das inserções após 8 culturas sequenciais com pelo menos 10 gerações cada. Apesar de crescer em lactose como única fonte de carbono e energia, a produção de β-galactosidase, o nível de floculação e a rapidez no consumo de lactose são inferiores à estirpe de referência.Recombinant DNA technology is a widely used tool in the production of proteins in heterologous systems, namely for high value proteins that are, naturally, produced in low quantities. This work aims to construct flocculent Saccharomyces cerevisiae strains with Aspergillus niger β-galactosidase stable production by using the repeated chromosomal δ sequences of the yeast as target sites for the lacA gene (coding for A. niger β-galactosidase) integration. Two different integration systems were used. One uses as dominant selection marker the G418 antibiotic resistance and allows for tandem integrations, while the other uses the URA3 selection marker and integration occurs at different sites with one single copy of the gene at each site. The lacA gene, flanked by ADH1 promotor and terminator, was cloned in both systems that were used in the transformation of S. cerevisiae NCYC869-wt (MatαFlo1) and S. cerevisiae NCYC869-A3 (MatαFlo1ura3) strains, respectively. In the first system different G418 concentrations were used raging from 0.2 to 1.5 g/l while in the other system 3 rounds of transformation using the same selection marker were made, once the URA3 gene is “pooped” out by recombination between flanking direct hisG repeats. In addition, transformants were selected for further studies based on the blue tonality of the colony. Due to the presence of X-gal in selective medium plates, a deeper blue colour of the colony indicated increased β-galactosidase activity. Both integration systems resulted in recombinant S. cerevisiae strains that grow on lactose. Independently of the selection marker used, different flocculation and β-galactosidase expression levels were observed. Overall, transformants obtained from the first system presented higher β-galactosidase extracellular production. The most interesting transformants were characterized genetically by Southern analyses. For the first system, one or two integration sites were observed, with tandem copies and 8 gene copies integrated in maximum, while for the second system, after 3 rounds of transformation and URA3 gene loss, 2 different sites were used for integration, corresponding to 2 gene copies. The best transformant obtained, belonging to the first system (selected with G418 1.5 g/l), has β-galactosidase activity in glucose comparable to the reference epissomal based plasmid strain (S. cerevisiae NCYC869-A3/pVK1.1) and reveals great integration stability after 8 sequential batch cultures with, at least, 10 generations each. Despite of growing on lactose as the only carbon and energy source, β-galactosidase expression level, flocculation level and lactose consumption time were lower than those obtained with the reference strain

Expression and production of recombinant frutalin in different expression systems and evaluation of its biomedical applications

Este resumo faz parte de: Book of abstracts of the Meeting of the Institute for Biotechnology and Bioengineering, 2, Braga, Portugal, 2010. A versão completa do livro de atas está disponível em: http://hdl.handle.net/1822/10968Frutalin is the alpha-D-galactose-binding lectin expressed in breadfruit seeds (Artocarpus incisa). This lectin may be used in cancer diagnostics/therapeutics due to its potential ability to recognise specific carbohydrates expressed in cancer cells membranes and/or cells surface receptors. However, frutalin extraction from plant seeds is a time-consuming process and typically results in a heterogeneous mixture of different natural isoforms. To overcome these limitations, frutalin was cloned and expressed in Pichia pastoris and Escherichia coli. Recombinant frutalin was detected in cultures of these microorganisms by SDS-PAGE and Western blot analysis. The higher recombinant frutalin yield was obtained in the P. pastoris expression system (up to 20 mg/L) [1]. Molecular and biological differences were found between each recombinant frutalin and native frutalin. Potential biomedical applications for native frutalin and recombinant frutalin produced in P. pastoris were studied. Recombinant frutalin demonstrated higher capacity than native frutalin to differentiate malign from benign human prostate diseases by immunohistochemistry (with a significant positive statistical correlation, P<0.00001), in spite of its lower carbohydrate-binding affinity [2]. In addition, native and recombinant frutalin showed an identical magnitude of cytotoxicity on HeLa cervical cancer cells growth (IC50=100 microgram/mL, 24 h), by inducing cell apoptosis and inhibiting cell proliferation and migration. Interaction studies conducted by confocal microscopy showed that native and recombinant frutalin were internalised and targeted to HeLa cell’s nucleus within 1 h of incubation. Therefore, frutalin with promising application in cancer diagnosis and therapy might be obtained from the recombinant P. pastoris expression system in alternative to its natural source. References [1] Oliveira C, Felix W, Moreira RA, Teixeira JA, Domingues L, “Expression of frutalin, an alpha-D-galactose-binding jacalin related lectin, in the yeast Pichia pastoris”, Prot. Exp. Pur. (2008) 60:188-193. [2] Oliveira C, Teixeira JA, Schmitt F, Domingues L, “A comparative study of recombinant and native frutalin binding to human prostate tissues”, BMC Biotechnol. (2009) 9:78

Study of the effects of High Hydrostatic Pressure (HHP) and Pulsed Light (PL) on BSA structure and hydrolysis

Non-thermal technologies, such as High Hydrostatic Pressure (HHP) and Pulsed light (PL), affect protein inducing modifications in its conformational structure. For this reason the hydrolysis reaction of the protein can be modulated if it is conducted in combination with these technologies which are able to change the availability of peptide bonds exposed to the enzymatic action. The aim is to study the effects of HHP and PL on the structure and the extent of hydrolysis reaction of a target protein: the Bovine Serum Albumin (BSA). BSA protein (5 mg/mL) in sodium phosphate buffer (50 mM, pH = 8) were treated with PL and HHP at different processing conditions, namely pressure level and treatment time in the case of HHP and treatment time and energy input in the case of PL. Structural modification of the protein solutions were analyzed by determining the sulphidrilic groups and the changes of the secondary structure. The effect of the two treatments on the hydrolysis degree (HD) at 37 °C was also evaluated by OPA method. Chymotrypsin and trypsin (E/S ratio = 1/10) were used to hydrolyze the BSA protein solutions. The hydrolysis was carried out in HHP assisted or PL assisted conditions or the protein solutions were treated with HHP or PL processes and immediately after hydrolyzed with the enzymes. Results obtained so far demonstrated that the two technologies tested are able to induce protein modifications and the occurrence and importance of this phenomenon depends on processing parameters causing protein unfolding, namely pressure level and number of pulses. When the maximum protein unfolding is obtained, higher HD values are detected. The highest HD value is obtained in HHP assisted hydrolysis with longer treatment time, and when, before undergoing hydrolysis, the PL treatment is applied to the solution placed at the higher distance from the lamp

Recombinant microbial systems for improved β-galactosidase production and biotechnological applications

β-Galactosidases (EC 3.2.1.23) constitute a large family of proteins that are known to catalyze both hydrolytic and transgalactosylation reactions. The hydrolytic activity has been applied in the food industry for decades for reducing the lactose content in milk, while the transgalactosylation activity has been used to synthesize galacto-oligosaccharides and galactose containing chemicals in recent years. The main focus of this review is on the expression and production of Aspergillus niger, Kluyveromyces lactis and bacterial β-galactosidases in different microbial hosts. Furthermore, emphasis is given on the reported applications of the recombinant enzymes. Current developments on novel β-galactosidases, derived from newly identified microbial sources or by protein engineering means, together with the use of efficient recombinant microbial production systems are converting this enzyme into a relevant synthetic tool. Thermostable β-galactosidases (cold-adapted or thermophilic) in addition to the growing market for functional foods will likely redouble its industrial interest.C. Oliveira and P. M. R. Guimaraes acknowledge support from Fundacao para a Ciencia e a Tecnologia (FCT), Portugal (grants SFRH/BDP/63831/2009 and SFRH/BDP/44328/2008, respectively)

Isolation and molecular cloning of γ-terpinene synthase gene from thymus caespititius

Poster session from thematic symposium 8

Delta multicopy integration for improved β-galactosidase production in recombinant Saccharomyces cerevisiae

The β-galactosidase industrial production is hampered by the high costs associated with its production and purification. One way to improve the overall productivity of galactosidase β-galactosidase fermentation system would be to use continuous high-cell-density systems. Among these, the ones that use flocculent cells are surely attractive due to its simplicity and low cost. We have previously reported the construction of a flocculent Saccharomyces cerevisiae strain secreting high levels of Aspergillus niger β-galactosidase. Due to the cell flocculation characteristics, the recombinant yeast may be used in a high-cell-density system operating in continuous mode. However, when operating at high dilution rates we have observed some plasmid instability which led to a decrease in the β-galactosidase production. With this work we aim at obtaining stable yeast transformants with at least the same β-galactosidase production level of the previously constructed strain1'1 (construction based on an epissomal plasmid) but with enhanced stability which would allow to increase the enzyme productivity in the continuous system. For that, the lacA gene from A. niger (coding to β-galactosidase) was integrated into the genome of the flocculent yeasts S. cerevisiae NCYC 869 and S. cerevisiae NCYC 869-A3 (ura) using integrative vectors with a G418 and ura3 marker, respectively. The repeated cromossomal δ sequences of the yeasts were employed as target sites for the integration. The S. cerevisiae NCYC 869 integrants were selected by resistance to the aminoglycoside G418 (0.2-1.5 g/I) while for the auxothrophic strain S. cerevisiae NCYC869-A3 the selection of integrants was made on minimal medium. Some transforming colonies that presented a deep blue tonality (due to the presence of the X-gal in the plates of selective medium) were randomly selected for growth in nonselective liquid media containing lactose or glucose. Different levels of β-galactosidase expression were observed independently of the selection marker used. For ones that presented more enzyme activity, expression levels of β-galactosidase, cell growth and substrate consumption were found to be similar with the previous y constructed strain (with a 2μ-based plasmid). Unexpectedly, the flocculation of the original strains was affected by the integration. The most flocculants were from transformation using the ura3 marker selection system and the second ones were from transformation using 1.5 g/I G418 as selective marker. Nevertheless, all transformants were less flocculent when compare with the original strain. Transformants genetic characterization by Southern analysis confirmed the multicopy tandem integration pattern. For the analysed transformants, one or two different integration sites were observed. For the most promising transformants, physiological and genetic characterization is being conducted in order to select for a new recombinant strain to be used in a continuous high-cell-density β-galactosidase producing system

Recombinant CBM-fusion technology : applications overview

Carbohydrate-binding modules (CBMs) are small components of several enzymes, which present an independent fold and function, and specific carbohydrate-binding activity. Their major function is to bind the enzyme to the substrate enhancing its catalytic activity, especially in the case of insoluble substrates. The immense diversity of CBMs, together with their unique properties, has long raised their attention for many biotechnological applications. Recombinant DNA technology has been used for cloning and characterizing new CBMs. In addition, it has been employed to improve the purity and availability of many CBMs, but mainly, to construct bi-functional CBM-fused proteins for specific applications. This review presents a comprehensive summary of the uses of CBMs recombinantly produced from heterologous organisms, or by the original host, along with the latest advances. Emphasis is given particularly to the applications of recombinant CBM-fusions in: (a) modification of fibers, (b) production, purification and immobilization of recombinant proteins, (c) functionalization of biomaterials and (d) development of microarrays and probes.Fundação para a Ciência e a Tecnologia (FCT), Portugal (grants SFRH/BDP/63831/ 2009 and SFRH/BPD/73850/2010, respectively). The authors thank the FCT GlycoCBMs Project REF. PTDC/AGR-FOR/3090/2012 — FCOMP-01- 0124-FEDER-027948, the FCT Strategic Project PEst-OE/EQB/LA0023/ 2013, and the Project “BioInd — Biotechnology and Bioengineering for improved Industrial and Agro-Food processes”, REF. NORTE-07-0124- FEDER-000028 Co-funded by the Programa Operacional Regional do Norte (ON.2 — O Novo Norte), QREN, FEDER