Contents 1 Placement of Oligoarginine Tail on Proteins 1.1 Hypothesis 1.2 Introduction 1.3 Method 1.4 Results

Placement of Oligoarginine Tail on Proteins

Hypothesis

The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein in vivo.

Introduction

The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting research on the project, we came upon data suggesting that arginine residues at the N-terminus of a protein would significantly reduce its half-life, and causing degradation of our protein before it can be moved into the microcompartment. We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.

Method

In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks BBa_K249005 and K249004 respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (BBa_K331023) or N-terminus (BBa_K331022) (and preceded by a ribosomal binding site – B0034) were synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (BBa_R0010) for constitutive expression of the fusion protein. This addition generated BioBricks BBa_K331031 and BBa_K331030 for the C-terminal tagged and N-terminal tagged YFP respectively. The BioBrick containing plasmid was transformed into Escherichia coli DH5cells. These cells were grown to an OD600 of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy. This dilution of cells was excited at 517nm, and the emission spectra was read from 522nm to 650nm. Fluorescence at 524nm (emission maxima of YFP) of control cells (Escherichia coli DH5), N-terminal tagged, and C-terminal tagged YFP were compared.

Results

N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.