XI Salão de Iniciação Científica – PUCRS, 09 a 12 de agosto de 2010

XI Salão de

Iniciação Científica PUCRS

Expression of recombinant bovine somatotropin (r-bST) in Escherichia coli cells.

Rafael Munareto1,2,3, José Eduardo Sacconi Nunes2,3, Jocelei Maria Chies3, Luiz Augusto Basso2, Gaby Renard3, Diógenes S. Santos1,2

1Faculdade de Farmácia, PUCRS, 2Centro de Pesquisa em Biologia Molecular e Funcional, InstitutoNacional de Pesquisa em Tuberculose, 3Quatro G Pesquisa & Desenvolvimento Ltda

Introduction

Bovine growth hormone (bGH) or bovine somatotropin (bST), is a hormone produced in the

pituitary gland of cattle. The bovine pituitary gland naturally secretes bST into the

bloodstream, which acts on receptors in the liver to produce Insulin-like Growth Factor 1

(IGF-1). These two hormones have many different effects in the body, including increasing

the metabolism of lipids for energy and helping to prevent mammary cell death. bST

administration to dairy cows increases milk production and improves the efficiency of milk

synthesis (Bauman, 1999). In November 1993, recombinant bovine somatotropin (r-bST) was

among the first agricultural biotechnology products to be approved by the U.S. Food and

Drug Administration (FDA). Since Brazil imports this product, the optimization of the

overproduction and purification of the r-bST represents an important step for the development

of new technologies for the national production of this protein. The aim of this study is to

construct and clone a synthetic bovine growth hormone gene with expression in E. coli cells

for further purification of the recombinant hormone.

Methods

Construction of the gene coding sequence of the bovine growth hormone:

For the construction of the gene, primers were designed based on the bovine growth hormone

sequence (DQ184480). The restriction enzyme sites for NdeI and BamHI were added at the 5´

and 3´ ends, respectively. The primers were assembled using the Pfu DNA Polymerase in

polymerase chain reactions by a method developed in our laboratory (PCT/Br 2006/000267)

(Renard, 2007).

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XI Salão de Iniciação Científica – PUCRS, 09 a 12 de agosto de 2010

Cloning and subcloning:

The constructed gene was cloned into the PCR®-Blunt (Invitrogen) cloning vector and

subcloned into the expression vector pET30a(+) (Novagen). The vector DNA was cleaved

with the restriction enzymes NdeI and BamHI. The clone that showed the expected insert size

was subjected to automatic sequencing confirming their authenticity and absence of

mutations.

Expression of bovine growth hormone:

Different E. coli cells were transformed by electroporation with the recombinant plasmid and,

as negative control, E. coli cells were transformed with the pET 30a(+) vector lacking the

insert. The r-bST was expressed in the insoluble fraction of BL21 (DE3) Star, C41 (DE3),

Rosetta-gami 2 (DE3), Rosetta (DE3), BL21 (DE3), and BL21 (DE3) NH strains.

Solubilization of inclusion bodies:

For solubilization of inclusion bodies the denaturing agents urea (2M, 4M, 6M and 8M) and

guanidinium chloride (2M, 4M and 6M) were tested.

Conclusion and Perspectives

The gene construction, cloning, and expression were successfully achieved. As the protein

was expressed in the insoluble fraction some solubilizing agents were tested. The treatment

with guanidinium chloride was unsuccessful while the treatment with urea 8M was able to

solubilize the inclusion bodies. These agents are commonly used for proteins expressed in

inclusion bodies form (Tsumoto et al., 2003). The next step in this study will be the protein

purification by liquid chromatography where different resins will be tested. This work is

important for the development of technologies for the national production of the recombinant

bovine growth hormone.

References Bauman, DE, Bovine somatotropin and lactation: From basic science to commercial application. Domes Anim Endocrinol 17 (1999), pp. 101–116. Renard, G., Fonseca, I.O., Chies, J.M., Basso, L.A. and Santos, D.S. (2007) Method for obtention of Chimeric Nucleotides Sequences and Chimeric Nucletide Sequence.WO2007/068068A2. TSUMOTO, K.; EJIMA, D.; KUMAGAI, I.; ARAKAWA, T. Practical considerations in refolding proteins from inclusion bodies. Protein Expression and Purification, v. 28, n. 1, p. 1-8, 2003.

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