Genetic engineering of novel protein polymers and their biosynthetic production in Escherichia coli and Pichia pastoris.

摘要

With an interest in developing environmentally triggered protein-based materials, we have studied the microbial production of a self-assembling β-sheet polymer, poly-EAK9, composed of the alternating amino acid repeat sequence, AEAEAKAK. Protein sequences of alternating hydrophilic (E = Glutamate, K = Lysine) and hydrophobic (A = Alanine) residues are known to adopt β-sheet structures in aqueous solutions, and previous work by Zhang and co-workers on ionic self-complementary peptides have demonstrated their ability to self-assemble into macroscopic membranes, fibrils, and gels upon the addition of monovalent metal ions. Through the use of recombinant DNA techniques, we have constructed a high molecular weight analogue of the EAK sequence to probe its self-assembly and mechanical properties for potential use in biomaterial applications, such as local drug delivery, tissue engineering, and biosensors.; A second class of protein-based materials, consisting of sequence disordered copolymers of leucine (L) and either serine (S), glutamate (E), or lysine (K) residues, were constructed and studied for expression in Escherichia coli. Statistically patterned protein polymers are of particular interest in the study of adsorption to multi-functionalized surfaces. Monte Carlo simulations and theoretical work by A. K. Chakraborty and co-workers suggest that disordered copolymers may preferentially adsorb to certain patterned surfaces when the statistics of the polymer sequence and the distribution of surface sites are related in a specific way. To probe such phenomena, the sequence statistics of disordered protein polymers could be studied for their effect on adsorption to similarly patterned surfaces, such as mixed SAMS consisting of functionalized alkanethiolates. Future development of synthetic systems that can mimic such recognition would have significant impact on applications ranging from high throughput nano-scale separation processes to viral inhibition and the development of sensors.; As de novo designed protein polymers diverge from natural protein sequences, heterologous gene expression can drop significantly. Several artificial proteins, including disordered poly-EL and poly-KL polymers, displayed poor production levels in Escherichia coli. Possible reasons for this are presented and improved production from a yeast expression system using Pichia pastoris is reported. Finally, future directions and applications of artificial protein polymers will be discussed. (Abstract shortened by UMI.)