Expanding the recombinant protein quality in Lactococcus lactis.

BACKGROUND: Escherichia coli has been a main host for the production of recombinant proteins of biomedical interest, but conformational stress responses impose severe bottlenecks that impair the production of soluble, proteolytically stable versions of many protein species. In this context, emerging Generally Recognized As Safe (GRAS) bacterial hosts provide alternatives as cell factories for recombinant protein production, in which limitations associated to the use of Gram-negative microorganisms might result minimized. Among them, Lactic Acid Bacteria and specially Lactococcus lactis are Gram-positive GRAS organisms in which recombinant protein solubility is generically higher and downstream facilitated, when compared to E. coli. However, deep analyses of recombinant protein quality in this system are still required to completely evaluate its performance and potential for improvement. RESULTS: We have explored here the conformational quality (through specific fluorescence emission) and solubility of an aggregation-prone GFP variant (VP1GFP) produced in L. lactis. In this context, our results show that parameters such as production time, culture conditions and growth temperature have a dramatic impact not only on protein yield, but also on protein solubility and conformational quality, that are particularly favored under fermentative metabolism. CONCLUSIONS: Metabolic regime and cultivation temperature greatly influence solubility and conformational quality of an aggregation-prone protein in L. lactis. Specifically, the present study proves that anaerobic growth is the optimal condition for recombinant protein production purposes. Besides, growth temperature plays an important role regulating both protein solubility and conformational quality. Additionally, our results also prove the great versatility for the manipulation of this bacterial system regarding the improvement of functionality, yield and quality of recombinant proteins in this species. These findings not only confirm L. lactis as an excellent producer of recombinant proteins but also reveal room for significant improvement by the exploitation of external protein quality modulators.

Increase in post-thaw viability by adding seminal plasma proteins to Sanmartinero and Zebu sperm / Incremento en la viabilidad espermática post-descongelación por la adición de proteínas del plasma seminal a semen de toros Sanmartinero y Cebú / Aumento da viabilidade espermática pós-descongelamento, com a adição de proteínas do plasma seminal de sêmen de touros das raças Sanmartinero e Zebu

Background: cryopreservation decreases sperm viability by approximately 50%. Objective: the objective of this study was to determine the effect of the addition of seminal plasma proteins on post-thawing sperm viability in Sanmartinero and Zebu semen. Methods: semen samples from 10 bulls of each breed were used, and seminal plasma was subjected to two-dimensional electrophoresis to establish the relationship between the relative amount of each protein spot and sperm viability. Then, seminal plasma was subjected to exclusion chromatography to separate the fraction containing these proteins. This fraction was added in doses of 0.5, 1.0, 1.5 and 2.0 mg, to 1 x 10(6). Sperm was thawed and incubated at 37 °C for 1 h to determine its effect on postthaw viability. Sperm were frozen using two media (citrate-fructose-yolk and Bioxcell®). Results: we found one protein spot (16.20 kDa, PI 5.5) in Sanmartinero seminal plasma that correlated (r = 0.64 p<0.001) with viability. This spot was found in 21-25 chromatography fractions. The percentage of post-thaw viable sperm increased 20% (p<0.05) at 1.0 and 1.5 mg of the fraction when sperm was frozen using citrate-fructose-yolk; it increased 25% (p<0.01) with 0.5 mg when it was frozen with Bioxcell® media. Addition of 0.5 mg of the fraction to semen cryopreserved with Bioxcell® resulted in a greater (p<0.05) percentage increase of viable sperm in Sanmartinero semen (23 ± 8.3%) compared with Zebu semen (6.0 ± 2.0%). Conclusions: these results show that seminal plasma proteins decrease cryopreservation damage in sperm. The effect depends on the cryoprotectant dose as well as the breed of bull.

Antecedentes: la criopreservación disminuye la viabilidad espermática por debajo del 50%. Objetivo: el objetivo de esta investigación fue determinar el efecto de la adición de proteínas del plasma seminal sobre la viabilidad espermática post-descongelación de semen de toros Sanmartinero y Cebú. Métodos: se colectó semen de 10 toros de cada raza, y el plasma seminal se sometió a electroforesis bidimensional, para establecer la relación entre la cantidad relativa de cada punto de proteína y la viabilidad espermática. Identificados dichos puntos, el plasma seminal se sometió a cromatografía de exclusión para separar la fracción que contenía estas proteínas. Esta se adicionó en dosis de 0,5, 1,0, 1,5 y 2,0 mg, a muestras de 1 x 10(6) espermatozoides, descongelados e incubados a 37 °C durante 1 hora, para determinar su efecto en la viabilidad post-descongelación. Los espermatozoides se congelaron usando dos medios (citrato-fructosa-yema y Bioxcell®). Resultados: se encontró un punto de proteína (16,20 kDa, punto Isoeléctrico 5,5) en plasma de toros Sanmartinero, que correlacionó (r = 0,64 p<0,001) con la viabilidad. Este punto de proteína se encontró en la fracción 21-25 de la cromatografía. El porcentaje de espermatozoides viables post-descongelación aumentó 20% (p<0,05) con dosis de 1 y 1,5 mg de la fracción, cuando los espermatozoides se congelaron en medio citrato-fructosa-yema; y 25% (p<0,01) con dosis de 0,5 mg cuando se congelaron en medio Bioxcell®. La adición de 0,5 mg de la fracción a semen descongelado previamente criopreservado en medio Bioxcell®, evidenció un incremento mayor (p<0,05) en el porcentaje de espermatozoides viables de semen de toros Sanmartinero (23 ± 8,3 %), que en semen de toros Cebú (6,0 ± 2,0%). Conclusiones: los resultados anteriores demuestran que las proteínas del plasma seminal disminuyen el daño en los espermatozoides por la criopreservación, y que el efecto de estas proteínas depende del medio de congelación, la dosis adicionada y la raza de los toros.

Antecedentes: a criopreservação diminui a viabilidade espermática abaixo de um 50%. Objetivo: o objetivo desta pesquisa foi determinar o efeito da adição de proteínas do plasma seminal na viabilidade espermática pós-descongelamento de sêmen de touros das raças Sanmartinero y Zebú. Métodos: coletou-se sêmen de 10 touros de cada raça, as amostras do plasma seminal foram submetidas à eletroforese bidimensional, para estabelecer a relação entre a quantidade relativa de cada ponto de proteína e a viabilidade espermática. Ao serem identificados os pontos, o plasma seminal também foi submetido ao processo de cromatografia por exclusão para separar a fração que continha as proteínas. A fração foi adicionada nas doses de 0,5, 1,0, 1,5 y 2,0 mg, amostras de 1 x 106 espermatozoides, em descongelamento e incubados à temperatura de 37 ° C durante 1 hora, para determinar o efeito na viabilidade pós-descongelamento. Os espermatozoides foram congelados utilizando dois meios (Citrato- frutose-gema e Bioxcell®). Resultados: encontrou-se um ponto de proteína (16,20 kDa, ponto Isoelétrico 5,5) no plasma de touro Sanmartinero, que correlacionou (r=0,64 p<0,001) com a viabilidade. Esse ponto de proteína foi encontrado na fração 21-25 da cromatografia. O percentagem de espermatozoides viáveis pós-descongelamento aumentou em 20% (p<0,05) nas doses de 1 y 1,5 mg da fração, quando os espermatozoides foram congelados em meio de citrato-frutose-gema; e 25% (p<0,01) com doses de 0,5 mg congelados em meio Bioxcell®. A adição de 0,5 mg da fração ao sêmen descongelado e previamente criopreservado em meio Bioxcell®, evidenciou um incremento maior (p<0,05) no percentagem de espermatozoides viáveis do sêmen de touros Sanmartinero (23 ± 8,3 %), do que em sêmen de touros zebu (6,0 ± 2,0%). Conclusões: os anteriores resultados demonstram que as proteínas do plasma seminal diminuem o dano nos espermatozoides pela criopreservação e que o efeito destas proteínas depende do meio de congelação, a dose adicionada e a raça dos touros.