RESUMEN
Recombinant protein expression for structural and therapeutic applications requires the use of systems with high expression yields. Escherichia coli is considered the workhorse for this purpose, given its fast growth rate and feasible manipulation. However, bacterial inclusion body formation remains a challenge for further protein purification. We analyzed and optimized the expression conditions for three different proteins: an anti-MICA scFv, MICA, and p19 subunit of IL-23. We used a response surface methodology based on a three-level Box-Behnken design, which included three factors: post-induction temperature, post-induction time and IPTG concentration. Comparing this information with soluble protein data in a principal component analysis revealed that insoluble and soluble proteins have different optimal conditions for post-induction temperature, post-induction time, IPTG concentration and in amino acid sequence features. Finally, we optimized the refolding conditions of the least expressed protein, anti-MICA scFv, using a fast dilution protocol with different additives, obtaining soluble and active scFv for binding assays. These results allowed us to obtain higher yields of proteins expressed in inclusion bodies. Further studies using the system proposed in this study may lead to the identification of optimal environmental factors for a given protein sequence, favoring the acceleration of bioprocess development and structural studies.
Asunto(s)
Clonación Molecular/métodos , Escherichia coli/genética , Antígenos de Histocompatibilidad Clase I/genética , Interleucina-23/genética , Anticuerpos de Cadena Única/genética , Secuencia de Aminoácidos , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Análisis Factorial , Expresión Génica/efectos de los fármacos , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/aislamiento & purificación , Humanos , Cuerpos de Inclusión/química , Interleucina-23/química , Interleucina-23/aislamiento & purificación , Isopropil Tiogalactósido/farmacología , Análisis de Componente Principal , Replegamiento Proteico , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/aislamiento & purificación , SolubilidadRESUMEN
BACKGROUND: Cancer cells are known to secrete the stress molecules MICA and MICB that activate cytotoxicity by lymphocytes and NK cells through their NKG2D receptor as a mechanism of immunological defense. This work was undertaken to evaluate if cancer cells can also express this receptor as a possible mechanisms of depletion of MIC molecules and thus interfere with their immune recognition. METHODS: Myelomonocytic leukemic (TPH-1 and U-937) and cervical cancer (CALO and INBL) cell lines were evaluated by Western Blot, ELISA, flow cytometry and immunocytochemistry to evaluate their capacity to express and secrete MICA and MICB and to be induced to proliferate by these molecules as well as to express their receptor NKG2D. Statistical analysis was performed by two-way ANOVA for time course analysis and Student's t-test for comparison between groups. Values were considered significantly different if p < 0.05. RESULTS: THP-1 and U-937 produce and secrete the stress MICA and MICB as shown by Western Blot of lysed cells and by ELISA of their conditioned media. By Western Blot and flow cytometry we found that these cells also express the receptor NKG2D. When THP-1 and U-937 were cultured with recombinant MICA and MICB they exhibited a dose dependent induction for their proliferation. CALO and INBL also produce MICA and MICB and were induced to proliferate by these stress molecules. By Western Blot, flow cytometry and immunocytochemistry we also found that these cells express NKG2D. CONCLUSIONS: Our novel results that tumor cells can simultaneously secrete MIC molecules and express their receptor, and to be induced for proliferation by these stress molecules, and that tumor epithelial cells can also express the NKG2D receptor that was thought to be exclusive of NK and cytotoxic lymphocytes is discussed as a possible mechanism of immunological escape and of tumor growth induction.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Leucemia Mieloide/metabolismo , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Medios de Cultivo Condicionados , Femenino , Antígenos de Histocompatibilidad Clase I/aislamiento & purificación , Antígenos de Histocompatibilidad Clase I/farmacología , Humanos , Leucemia Mieloide/patología , Monocitos/metabolismo , Neoplasias del Cuello Uterino/patologíaRESUMEN
HLA-G is a nonclassical class I MHC molecule of unknown function expressed on human trophoblast. The level of polymorphism at the HLA-G locus is of considerable importance, since the paternally inherited gene product is exposed to the maternal immune system during pregnancy. However, previous studies of HLA-G polymorphism using genomic DNA samples have produced conflicting results. Our aim was to investigate polymorphism in trophoblast HLA-G mRNA from pregnancies in ten Caucasian and twelve Afro-Caribbean women by RT-PCR. A similar PCR protocol was also applied to umbilical cord blood genomic DNA from two Caucasian and two Afro-Caribbean neonates. Caucasian cDNA yielded only two different sequences: G*01011, and one containing a previously reported synonymous substitution. Afro-Caribbean samples yielded these sequences as well as one previously reported conservative (leucine-to-isoleucine) substitution. PCR amplification from genomic DNA samples from both populations using previously published primer pairs generated sequences containing multiple substitutions, many of which were nonsynonymous. More than two sequences were produced from genomic DNA from each individual. In contrast, amplification from the same genomic DNA using new primers compleentary to exons of the HLA-G gene yielded the same few sequences generated from cDNA. These results suggest that polymorphism at the HLA-G locus is extremely limited in Caucasian and Afro-Caribbean populations. This suggests that spurious polymorphism has been reported in African Americans due to the use of intron-complementary PCR primers on genomic DNA samples. The monomorphic nature of HLA-G may allow trophoblast to carry out the immunological functions of class I-bearing tissues without compromising successful pregnancy.(Au)
Asunto(s)
Femenino , Humanos , Recién Nacido , Antígenos de Histocompatibilidad Clase I/genética , Antígenos HLA/genética , Polimorfismo Genético , ARN Mensajero/aislamiento & purificación , África/etnología , /genética , Corion/química , ADN/química , Cartilla de ADN/química , ADN Complementario/química , Exones , Sangre Fetal/química , Feto , Amplificación de Genes , Antígenos de Histocompatibilidad Clase I/aislamiento & purificación , Antígenos HLA/aislamiento & purificación , Intrones , /genética , Embarazo , Región del Caribe/epidemiologíaRESUMEN
Major histocompatibility complex expression of activated peripheral blood lymphocytes of captive African green monkeys from Barbados and from Africa were analyzed biochemically; class I molecules by one-dimensional isoelectric focusing and class II DR molecules by one-dimensional nonequilibrium pH gradient electrophoresis. Much less diversity was observed in the major histocompatability molecule expression of the African green monkeys of Barbados than in the African cohort.