Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii

Abstract

SummaryRecombinant proteins are widely used today in many industries, including the biopharmaceutical industry, and can be expressed in bacteria, yeasts, mammalian and insect cell cultures, or in transgenic plants and animals. In addition, transgenic algae have also been shown to support recombinant protein expression, both from the nuclear and chloroplast genomes. However, to date, there are only a few reports on recombinant proteins expressed in the algal chloroplast. It is unclear whether this is because of few attempts or of limitations of the system that preclude expression of many proteins. Thus, we sought to assess the versatility of transgenic algae as a recombinant protein production platform. To do this, we tested whether the algal chloroplast could support the expression of a diverse set of current or potential human therapeutic proteins. Of the seven proteins chosen, >50% expressed at levels sufficient for commercial production. Three expressed at 2%-3% of total soluble protein, while a forth protein accumulated to similar levels when translationally fused to a well-expressed serum amyloid protein. All of the algal chloroplast-expressed proteins are soluble and showed biological activity comparable to that of the same proteins expressed using traditional production platforms. Thus, the success rate, expression levels, and bioactivity achieved demonstrate the utility of Chlamydomonas reinhardtii as a robust platform for human therapeutic protein production.

N-Glycosylation of the 66-kDa Cell-Wall Glycoprotein of a Red Microalga

“…A growing number of scientists around the world are building a novel assortment of pharmaceutical products using algae as cell factories [64][65][66]. However, although they are well suited to the large-scale production of recombinant proteins, algae have not been extensively utilized for protein expression [66][67]. There are a number of advantages in cultivating algae as a platform for producing therapeutic proteins.…”

“…A stable chloroplast transformation system [62], and recently, a nuclear transformation system, have been developed [63], the latter of which has paved the way for the expression of foreign genes in red algae and has far-reaching biotechnological implications. A growing number of scientists around the world are building a novel assortment of pharmaceutical products using algae as cell factories [64][65][66]. However, although they are well suited to the large-scale production of recombinant proteins, algae have not been extensively utilized for protein expression [66][67].…”

The microalgaChlamydomonas reinhardtiias a platform for the production of human protein therapeutics

“…Many of the proteins that have been quantified have been shown to accumulate as high as 2-20% of total soluble protein (TSP). [14][15][16] Importantly, the chloroplast contains the proper machinery to form disulfide bonds and assemble chloroplast. 16 VEGF and HMGB1 were purified from algal lysates and tested for bioactivity.…”

“…1). 16 The promoter regions are responsible for initiating transcription, while 5' UTRs function to regulate both mRNA stability as well as translation. 19 The 3' UTRs appear to influence mRNA processing and stability, [20][21][22][23] but may also interact with 5' UTRs to influence translation.…”

“…Thus, we sought to challenge the microalga chloroplast expression system with seven recombinant genes encoding for a diverse set of current or potential human therapeutic proteins (table 1). 16 The seven recombinant genes were codon-optimized for C. reinhardtii chloroplast codon bias and tagged with a FLAG tag to enable detection by western blot and for purification. Each was cloned into three distinct chloroplast transformation vectors: (1) pD1-KanR under the control of the psbA promoter and 5' and 3' untranslated regions (UTRs) with an integration site at the psbA locus resulting in replacement of the endogenous psbA gene, (2) the N-terminal tagging vector pD1-KanR-SAA, which is identical to requirements are minimal and thus it is inexpensive to culture and relatively easy to keep sterile.…”