[Biosynthesis and purification of human beta2-adrenergic receptor expressed in methylotrophic yeast Pichia pastoris].

Human beta2-adrenergic receptor is one of the most studied G-protein-coupled receptors. It plays a key role in autonomic nervous system and is a drug target in cardiovascular and pulmonary diseases. Despite the fact that its crystal structure was revealed, a physiological role and molecular mechanisms of its action remain largely unknown. We designed the construct pVR2ADRH, which contained the gene for human beta2-adrenergic receptor with a polyhistidine tag C-terminal extension. The recombinant DNA was used for transformation of the GS115 strain of Pichia pastoris. The heterologous expression level obtained was about 20 mg/l. The receptor was extracted from membrane fraction and was purified by metal-affinity and ion-exchange chromatography. The active receptors were isolated by alprenolol-sepharose CL-4B. The resulting level of purified human beta2-adrenergic receptor was approximately 1 mg per liter of culture. The homogeneity of the protein sample was confirmed by a dynamical light scattering analysis of the receptor's micellar solution.

[Cloning and expression of variants of the glutaryl-7-aminocephalosporic acid acylase of the bacterium Brevundimonas diminuta in Escherichia coli cells].

The gene coding for glutaryl-7-aminocephalosporic acid acylase (Gl7ACA acylase) of the bacterium Brevundimonas diminuta (BrdGl7ACA), a commercial enzyme widely used in modem biocatalytic technologies for manufacture of b-lactam antibiotics, was cloned. Efficient expression systems for producing a "native" recombinant BrdGl7ACA and its analogs modified by attaching affinity groups--the chitin-binding domain of chitinases A1 and hexahistidine sequence--were designed. It was demonstrated that both the recombinant hybrid proteins and the native Gl7ACA acylase produced in E. coli cells underwent a correct autoproteolytic processing with generation of functionally active enzymes and could be isolated with a high yield using one-step affinity chromatography.

Intraspecific heterogeneity of the Vicia faba mitochondrial genome: evidence for multiregional rearrangements in the mitochondrial chromosome associated with coxII-orf192 chimeric gene formation.

Previous RFLP-analysis of mtDNA isolated from different lines and cultivars of Vicia faba with respect to variability of the coxII gene revealed two types of mitochondrial genome: one with a normal coxII gene and the other with both normal coxII and chimeric coxII-orf192 genes. In this study we analyzed other regions of these two types of mitochondrial genome and found significant differences in the arrangement of regions around the coxII, coxIII, cob, rrn26 and atpA genes. More detailed analysis of the rrn26 and atpA gene regions showed that these genes are associated with recombinationally active repeats. Restriction maps of the rrn26 and atpA gene regions in different recombinative variants are presented.

Characterization of Vicia faba mitochondrial genomes with normal coxII and coxII-orf 192 chimeric genes.

Southern hybridization analysis of mitochondrial genomes from different lines and cultivars of Vicia faba, with respect to variability of the coxII gene sequence, revealed two predominant types of mitochondrial genomes. The type I mitochondrial genome contained the coxII gene sequence in a 6.5-kb BamHI fragment. Type II had two copies of the coxII sequence: the first in a 6.5-kb and the second in a 2.6-kb BamHI fragment. The second copy was represented by a coxII-orf192 chimeric gene. We found several pure lines with type I and type II mitochondrial genomes. Each type of genome was stably inherited. No chimeric gene was found in mitochondria of the male-sterile line cms447. Nucleotide sequences of Vicia faba mitochondrial DNA (mtDNA) containing normal and chimeric coxII genes are presented. The sequence of the normal coxII gene was compared to the coxII gene from mitochondria of Pisum sativum. The similarity of nucleotide sequences and of predicted amino acid sequences between these two genes was more than 98%. A very high similarity between transcription initiation and termination signals was also observed. The sequence of the chimeric gene was characterized at the 5' end by the almost complete sequence of the normal coxII gene, up to the fifth nucleotide before the termination codon. The 3' end of the chimeric gene was represented by the 3' part of an orf previously called orf128+. The full size of this orf was 576 nucleotides, and the full size of the predicted polypeptide was 192 amino acid residues. Therefore, this orf can be finally called orf192. Northern hybridization analysis showed that orf192 was actively transcribed into a 1.4-kb transcript. The chimeric gene was also transcribed into a minor transcript of about 3 kb. Comparative analysis of the normal coxII gene and orf192 supported the suggestion that the chimeric gene resulted from nonhomologous recombination.