Identification, cloning and expressions of proteases from a cold adapted organism Aliivibrio salmonicida

Abstract

The work presented in this thesis provides an overview of molecular and structural biology projects and their related problems. The problems arises during these projects are common for most of the target proteins, with varying intensity of severity. The realization and identification of these problems and selection of their correct, best possible solution is an imperative step that might lead to achievement of the goals. This project has started with the survey of a cold adapted bacterial genome for special kind of enzymes ‘Proteases’ that can hydrolyze other proteins. Working with these kinds of proteins has also increased the set of problems caused by instability due to cold adaptation and autolysis phenomenon related to proteases. Eight different proteases were chosen as targets, to be cloned, expressed, characterized and structurally resolved. The initial step was the bioinformatics analysis of chosen target that was essentially important to identify the function of domain in any chosen target. This identification helped making the decision to eliminate the unnecessary domains that can influence the overall success of the project. As one of our selected target TVS4041 (trypsin) has suffered the problems in expression, solubility and purification, due to the accumulation of hydrophobic residues in exposed C-terminal tail. In this research project we have attempted, two type of cloning techniques; Gateway cloning and traditional restriction digestion cloning. Gateway™ cloning technology was proved good for reliability, accuracy and importantly for the facilitation of switch-ability between expression vectors and expression host. This particular system could also try to be conserve, for time and efforts by ‘One-Tube BP and LR Gateway™ Reaction’. On the other hand traditional restriction digestion cloning for TVS4041 was also useful for direct one step transformation toward expression host but, with disadvantage of lack of switch-ability for different vectors and expression host. From the three expressed targets LexA, HslV and TVS4041 two were selected for extended expression trials for increased yield and solubility. Both selected target TVS4041 and LexA, were subjected to two different solubility enhancing techniques, these were varying expression condition and solubility tag attachment. TVS4041 was facing the problem of insolubility with increasing time of expression. Different expression conditions were tested and resulted in lower pH, low salt and reduced time of expression conditions for highest solubility. It was also suggested that lower pH (6) and absence of salt could be ideal during purification since in these condition, TVS4041 will be relatively inactive. Furthermore it is suggested that detected, insolubility residing C-terminal tail should be clipped and re-clone for prevention of aggregation during purification. In order to lowering the purification efforts and cost, it is suggested that methods should be experimentally evaluated for secretion of this protein into the media. The experimental work conducted with LexA expression in conjunction to different fusion tags resulted in order of Gb1>NusA>Z=Trx>MBP>6xHis, which was different then the results obtain from other proteins from same organism. These results confirm the complex nature of protein and prove that different protein behave uniquely in response to different fusion tags, irrespective of their belonging with same organism (Braud, Moutiez et al. 2005).