Purification of Regucalcin from the Seminal Vesicular Fluid A Calcium Binding Multi-Functional Protein

By: Ángela María Callejas

Ana Isabel Valencia Gómez MOLECULAR BIOLOGY.

INTRODUCTION

INTRODUCTION

Seminal fluid→ Grayish white bodily fluid.→ Contains sperms which can fertilize the female ova, approximately 40-600 million→ Volume of semen in an ejaculation: 2-5 ml.

The semen comprises of:• Fructose• Ascorbic acid• Zinc• Cholesterol• Protein• Calcium• Chlorine• Blood group antigens• Citric acid

• Magnesium• Vitamin B12• Phosphorus• Sodium• Potassium• Uric acid• Lactic acid• Nitrogen• DNA

CALCIUM.

→ Ion.→ 99% in the bone as crystals, 1% in LEC.→ Nerve transmission> Liberation NT, and hormonal secretion→ Neuromuscular junction> Acetyl hill.→ Hemostasis and coagulation.

Required For vascular contraction

and vasodilation

INTRODUCTION

REGUCALCINA. (RGN)

Calcium binding protein.Anti-apoptotic,anti-

prolific and anti-oxidative functions.

Maintenance of intracellular calcium

homeostasis.

-Can mediate cell death and proliferation by

modulating DNA synthesis and fragmentation.

-Involved in the synthesis of L-ascorbic acid that

scavenges the free radicals.

INTRODUCTION

-Inhibition of cell divsion and apoptotosis

suggestingits role in maintaining

prostate cancer.

Is mainly secreted from Sertoli cells: creates the

ideal conditions for spermatogonialdevelopment

Androgen target gene in rat testis, play a role in

spermatogenesis

REGUCLACIN is in:

Glandular epitelium of the bulbouterine and prostate gland

She half the homeostasis of

CALCIUM

SEMINIFEROUS TUBULAR FLUID

produced by: Sertoli cells

MALE REPRODUCTIVE TRACT

RELATIONSHIP

GENERAL OBJECTIVE.

The objective of the present study is to develop an immuno- affinity methodology to purify RGN from

seminal vesicular fluid.

MATERIALES Y METODOS

PCR→ Objetivo : Amplificación directa de un gen o fragmento de DNA o indirecta de un RNA→ La técnica se basa en la amplificación in vitro, incremento geométrico del numero de copias

de una secuencia particular de DNA. • Se deben aplicar los siguientes conceptos:1. Desnaturalización de DNA2. Hibridación de la hebra sencilla con un primer3. Replicación de la hebra sencilla por una DNA polimerasa a partir del primer.

variaciones del método: RT-PCRRAPDsPCR in situPCR-RFLPSSCPEntre otros.

MATERIALES Y METODOS

MATERIALES Y METODOS

DNA RECOMBINANTE.

→ El DNA se puede romper y unir con fragmentos de la misma especie o de una diferente.

Es necesario cortar (endonucleasa) y unir (ligasas) el DNA.• Las endonucleasas (enzimas de

restriccion): rompen la doble cadena de DNA al reconocer una secuencia especifica de bases. El rompimiento es especifico y reproducible.

• Las ligasas unen dos cadenas de DNA punta con punta.

MATERIALES Y MÉTODOS

Western blot o inmunoblot:

→ Proteínas son separadas por electroforesis, por medio de la técnica SDS-PAGE poliacrilamida gel electroforesis.

→ Luego se transfieren a un filtro→ Incubar con anticuerpos → El anticuerpo unido al filtro se detecta

de varias maneras, identificando la proteína contra la cual esta dirigido el anticuerpo.

Cromatografía SDS-PAGE.

Electroforesis en gel de poliacrilamida con dodecilsulfato sódico.

SDS desnaturaliza las proteínas, eliminando sus estructuras secundaria y

terciaria.

Fraccionamiento que obedece a: diferencia de peso; longitud de la cadena, y la forma de

la proteína.

Corriente eléctrica y migración

Gel de poliacrilamida

Tinción: Coomassie blue o tinción

argéntea

Reacción antígeno-anticuerpo

RESULTADOS

PCR

WESTERN BLOT

SDS-PAGE

SDS-PAGE

917 Pb

1.10h post Inducción2. Células no inducidas

1. Purificada

RESULTADOS

SDS- PAGE WESTERN BLOT 1. No escindida (51KDa) 2. Escindida (33KDa)

1. rRGN, con AntiRGN

RESULTADOS

WESTERN BLOT SDS- PAGE

1. Antes de tratamiento

2. Tratado3. Comprimidos

28KDa/ 34KDa/ 51KDa

DISCUSSIONAUTHOR SAID YES/NO

-Xie Z, Schendel S, Matsuyama S, Reed JC

-Wang CK, Lebowitz J, Cheung HC

- Laurentino S, Gonc¸alves J, Cavaco JE, Oliveira PF, Alves MG, de Sousa M, Barros A, Socorro S

• The elutes in SDS–PAGE showed a single band at 68 kDa in Coomassie R-250 staining. The acidic pH, used for elution of the protein from the IAC has been speculated to facilitate a conformational change in the protein favoring the dimerisation.

• This is the first report of the presence of RGN in the seminal vesicular fluid. Similar isoforms of RGN are reported in rat liver tissue.

AUTHOR SAID YES/NO

-Parrish JJ, Susko-Parrish JLGJ

-Arun P, Aleti V, Parikh K, Manne V, Chilukuri N

• Further, this protein had been confirmed as RGN by Liquid Chromatography Mass Spectrometry/Mass Spectrometry and has an average molecular weight of 33,878.07 Da suggesting that the 68 kDa protein is a dimeric form of RGN.

• A 917 bp PCR product had been amplified, including primer flanking sequence of 36 bp and actual coding 881 bp DNA sequence of buffalo RGN. The amplified buffalo RGN gene contained an open reading frame of 294 amino acids, which is 5 amino acids truncated from N-terminal-portion of the native RGN. The formation of low molecular weight isoforms of RGN having 28 and 24 kDa by proteolytic cleavage at the N-terminal end is reported.

DISCUSSION

CONCLUSION

→Proteins are responsible for the proper functioning of the human body. In this article the importance of RGN was exhibited, because it has a very important role in the homeostasis of calcium, control of apoptosis and anti-oxidative effect.

→This protein may help to control prostate cancer for their actions in inhibition of cell cycle and apoptosis.

→The authors Demonstrated that RGN is in vesicle seminal fluid by the utilization of PCR, western blot and SDS PAGE and the confirmation with liquid chromatography mass spectrometry.

→Being the regucalcin in the seminal fluid and have important functions could be used in a future synthetically for realization of drugs for the treatment of cancer especially the male reproductive tract.

CONCLUSION

• http://www.drelist.com/composition-of-human-semen/• http://www.news-medical.net/health/What-is-Semen.aspx• http://www.ncbi.nlm.nih.gov/pubmed/6151055• http://lymediseaseguide.org/es/test-accuracy-western-bl

ot• Martínez Sánchez , Lina María; Vargas Grisales, Natalia;

Cardona Mejía, Laura; Osorio Ospina, Felipe; Ramírez Pulgarin , Sergio. (29 de Julio de 2015). Biología Molecular. En Biología molecular (octava edición) (pág. 160-201). Medellín: Editorial Universidad Pontificia