Spinach chloroplast cpn21 co-chaperonin possesses two functional domains fused together in a toroidal structure and exhibits nucleotide-dependent binding to plastid chaperonin 60.

Chloroplasts contain a 21-kDa co-chaperonin polypeptide (cpn21) formed by two GroES-like domains fused together in tandem. Expression of a double-domain spinach cpn21 in Escherichia coli groES mutant strains supports growth of bacteriophages lambda and T5, and will also suppress a temperature-sensitive growth phenotype of a groES619 strain. Each domain of cpn21 expressed separately can function independently to support bacteriophage lambda growth, and the N-terminal domain will additionally suppress the temperature-sensitive growth phenotype. These results indicate that chloroplast cpn21 has two functional domains, either of which can interact with GroEL in vivo to facilitate bacteriophage morphogenesis. Purified spinach cpn21 has a ring-like toroidal structure and forms a stable complex with E. coli GroEL in the presence of ADP and is functionally interchangeable with bacterial GroES in the chaperonin-facilitated refolding of denatured ribulose-1,5-bisphosphate carboxylase. Cpn21 also inhibits the ATPase activity of GroEL. Cpn21 binds with similar efficiency to both the alpha and beta subunits of spinach cpn60 in the presence of adenine nucleotides, with ATP being more effective than ADP. The tandemly fused domains of cpn21 evolved early and are present in a wide range of photosynthetic eukaryotes examined, indicating a high degree of conservation of this structure in chloroplasts.

Stable expression plasmid for high-level production of GroE molecular chaperones in large-scale cultures.

A stable expression plasmid has been developed to overproduce the Escherichia coli GroES and GroEL molecular chaperones in large-scale cultures. This was achieved by cloning the groE operon under the transcriptional control of a bacteriophage T7 promoter to achieve regulated expression. Isopropyl-beta-D-thiogalactopyranoside (IPTG) induction of a lacUV5 regulated chromosomal copy of T7 gene 1, encoding viral RNA polymerase, resulted in high-level expression of the groE operon from a multicopy plasmid. Induced cells harboring the pT7groE expression plasmid accumulated GroEL to levels of 30% total cell protein, and GroES to 4-5%. Both overproduced proteins were recovered primarily from the soluble fraction of lysed cells. The T7 expression plasmid was significantly more stable than other groE expression plasmids tested during scale-up experiments, and could be used successfully for large-volume cultures of up to 200 l. Strain stability was greatly improved, compared to rich media, when cells were grown in a supplemented minimal medium.