Abstract
Streptococcus pneumoniae is a gram-positive bacterium, causative agent of many diseases, such as pneumonia, otitis and sinusitis. Vaccination is the most effective means of preventing infections caused by this bacterium, being the conjugated vaccine, in which the capsular polysaccharide (PS), main bacterial virulence factor, is covalently bonded to a carrier protein, the more recommended.The usage of a surface antigenic protein from the bacteria as the carrier protein can increase the capacity of immunization of the vaccine, minimizing the required quantity of PS, and therefore, production costs. Due to its antigenic features, protein PspA was selected for the production of a novel pneumococcal conjugated vaccine in the thematic project under development "Antipneumococcal conjugated vaccine: studies about the viability of a polysaccharide-pneumococcus surface protein A vaccine" (08/05207-4), coordinated by Dr. Martha Massako Tanizaki, from the Biotechnology Center of Butantan Institute.For obtaining the PspA protein, gram-negative bacteria Escherichia coli, widely used for the production of recombinant protein due to the ease of genetic manipulation and cultivation to high cell densities, was chosen. However, the production of a protein strange to the cell triggers stress responses that affect cell morphology and metabolism, such as the appearance of elongated cells during the induction phase, already reported in literature. In high cell density cultures, commonly used for protein production, cellular differentiation may contribute to a viscosity raise, affecting mainly the oxygen transfer.The aim of this study is to evaluate the influence of induction conditions and the accumulation of the interest protein on the cell, especially on its morphology.The experiments will be conducted in a shaker incubator, at 300 rpm, with two strains of E. coli (BL21(DE3) e M15), transformed with the plasmids pET37b+ and pQE30, respectively, for the expression of the proteins PspA4Pro and PspA94-PdT. IPTG and lactose will be evaluated as inductors, under moderate (27ºC) and accelerated (37ºC) conditions, in defined and complex culture media. For the lactose, different induction strategies will also be evaluated: pulse or gradual (autoinduction).The experiments will be monitored by measurements of cell concentration (optical density and dry mass), plasmid retention (plating in solid medium with and without antibiotic and colonies counting), protein concentration (Bradford method and electrophoresis gel densitometry), and glycerol, glucose, lactose and organic acids concentration (HPLC). The monitoring of the morphology will be made by observation of Gram stained slides in an optical microscope equipped with a digital camera, followed by the acquisition of digital images, which will be binarized (software ImageJ) and processed through a MatLab developed routine for the estimation of length and diameter of the cells. Culture conditions which favor cellular differentiation and/or protein synthesis will be selected for obtaining samples of cellular suspension for subsequent analysis in scanning electron microscope and more detailed characterization. The selected culture conditions will be further reproduced in bench bioreactor cultures for more detailed studies of the protein accumulation pattern and its influence in cell morphology.With the proposed studies, we hope to integrate the knowledge related to morphological and metabolic changes observed during heterologous production of the protein by E. coli and, hereby, to implement strategies for on-line modulation of induction.
|