Phycocyanin Fusion Constructs for Heterologous Protein Expression Accumulate as Functional Heterohexameric Complexes in Cyanobacteria

Overexpression of heterologous proteins from plants, bacteria, and human as fusion constructs in cyanobacteria has been documented in the literature. Typically, the heterologous protein P of interest is expressed as a fusion with the abundant CpcB beta-subunit of phycocyanin (PC), which was placed in the leader sequence position. The working hypothesis for such overexpressions is that CpcB*P fusion proteins somehow accumulate in a soluble and stable form in the cytosol of the cyanobacteria, retaining the activity of the trailing heterologous P protein of interest. The present work revealed a substantially different and previously unobvious picture, comprising the following properties of the above-mentioned CpcB*P fusion constructs: (i) the CpcB*P proteins assemble as functional (alpha,beta*P)(3)CpcG heterohexameric discs, where alpha is the CpcA alpha-subunit of PC, beta*P is the CpcB*P fusion protein, the asterisk denotes fusion, and CpcG is the 28.9 kDa PC disc linker polypeptide CpcG1. (ii) The (alpha,beta*P)(3)CpcG1 complexes covalently bind one open tetrapyrrole bilin alpha-factor per alpha-subunit and two bilins per beta-subunit. (iii) The (alpha,beta*P)(3)CpcG1 heterohexameric discs are functionally attached to the Synechocystis allophycocyanin (AP) core cylinders and efficiently transfer excitation energy from the assembled (alpha,beta*P)(3)CpcG1 heterohexamer to the PSII reaction center, enhancing the rate of photochemical charge separation and electron transfer activity in this photosystem. (iv) In addition to the human interferon a-2 and tetanus toxin fragment C tested in this work, we have shown that enzymes such as the plant-origin isoprene synthase, beta-phellandrene synthase, geranyl diphosphate synthase, and geranyl linalool synthase are also overexpressed, while retaining their catalytic activity in the respective fusion construct configuration. (v) Folding models for the (alpha,beta*P)(3)CpcG1 heterohexameric discs showed the recombinant proteins P to be radially oriented with respect to the (alpha,beta)(3) compact disc. Elucidation of the fusion construct configuration and function will pave the way for the rational design of fusion constructs harboring and overexpressing multiple proteins of scientific and commercial interest.

Automated summary

In this study, the authors revealed new properties of CpcB*P fusion constructs, elucidated the configuration and function of the fusion constructs, and showed examples of overexpressed enzymes in the fusion construct. These findings are significant for the rational design of fusion constructs harboring multiple expressed proteins.