Production of protein‐based polymers in Pichia pastoris

Abstract

A long-sought objective in material science is the development of polymers with controlled monomer sequence [1, 2]. Although progress has been made in synthetic chemistry [3], the level of control evident in natural sequential polymers such as DNA and proteins is unparalleled. These biological macromolecules feature a defined molecular size, as well as a controlled sequence of the nucleotide or amino acid monomers. Proteins fold into a three-dimensional structure defined by their primary sequence, resulting in unique properties. From 20 different amino acid monomers, nature has created an awe-inspiring wealth of different proteins, including enzymes, antibodies, and peptide hormones, as well as nonbioactive proteins with a structure-forming, rheological, or colloidal function. The latter category includes proteins such as collagen and elastin that fulfill a major role in the structure of various tissues, and silks used in animal architecture such as silkworm cocoons and spider webs (for more information see Section 9.1 of the General discussion). These proteins typically feature highly repetitive sequences with biased amino acid composition, and can often reversibly self-assemble into supramolecular structures through the formation of noncovalent bonds. The natural materials derived from them display remarkable toughness, elasticity, and other properties that have inspired material scientists to mimic them using modern protein engineering. These so-called protein-based polymers, or protein polymers for short, are produced as heterologous proteins in a suitable host, just like enzymes and other proteins, although their highly repetitive sequence, biased amino acid composition, and physicochemical properties do present additional difficulty. The genes encoding natural protein polymers are sometimes used, but more often genes are synthesized that encode simplified mimics, or even completely de novo-designed protein polymers [4-6]. Multifunctional block copolymers can be prepared by combining different polymer types into one molecule [1, 2, 7-9]. Ever since the pioneering work by Cappello and Ferrari of Protein Polymer Technologies [10, 11], Escherichia coli has by far been the most widely used production host for protein polymers. This workhorse of protein engineering is genetically very well accessible, and offers fast growth and efficient low-cost production [12]. Several other hosts have been used for the production of protein polymers, including plants, insect cells, transgenic animals, Aspergillus nidulans, Saccharomyces cerevisiae,

Mammalian cell lines used in bioprocessing

“…Moreover, besides removing the hypermannosylation gene (OCH1), glycosyltransferase and glycosidase genes are also transferred to manufacture the expected glycoprotein in another platform (16). The second most striking yeast expression system is based on Pichia pastoris, which can secrete adequately folded and active proteins, sustaining lower protein glycosylation and reaching high cell densities (17,18). Since N-linked glycosylations are different in higher eukaryotes, some yeasts have been genetically modified to carry out human-like N-linked glycosylation (19).…”

One-step heating strategy for efficient solubilization of recombinant spider silk protein from inclusion bodies

“…Additionally, a variety of heterologous expression hosts have also been attempted to produce recombinant spidroins, e.g. bacteria, yeast, plants, mammalian cells, and transgenic animals, each with its own pros and cons in terms of cost, manipulation, expression levels and contaminations [ 22 – 24 ]. The most widely used expression system is Escherichia coli ( E. coli ) owing to the most efficient, simple manipulation and cost-efficient production suitable for large-scale production [ 20 , 22 , 25 ].…”

“…Proteins extracted in presence of organic acids normally have to pass an affinity or ion exchange column, which is a long-lasting time and high cost process [ 29 ]. Besides, expression of recombinant spidroins has often resulted in insoluble IB formation [ 24 , 30 ], however, in some cases forming of IBs is advantageous as IBs are easily isolated with high yield and purity. Traditionally, protein solubilization from IBs is often achieved under harsh conditions, i.e.…”

Expanding Canonical Spider Silk Properties through a DNA Combinatorial Approach

“…Recent advances in genetic engineering have led to increased insight into silk proteins and the structural organization of spider-silk-encoding genes [ 17 , 18 ]. Hence, more research focused on the bioengineering technology to develop artificial spider silks comparable to natural spider silks mostly use bacteria [ 11 , 15 ], while yeast [ 19 , 20 ], mammalian cells [ 21 ], plants [ 22 ] and insect cells are also used as platforms. However, the cloning of spider silk genes presents both challenges and opportunities due to the highly repetitive nature of the native proteins known as spidroins [ 8 , 23 ].…”