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Preparation and characterization of bovine growth hormones produced in recombinant Escherichia coli.

ABSTRACT: Two analogues of bovine growth hormone (BGH) have been produced in Escherichia coli by recombinant DNA techniques. In analogue Delta-1, the N-terminal alanine residue of the full-length bovine sequence is replaced by methionine. In analogue Delta-9, which is expressed at much higher levels than is Delta-1, the full-length bovine sequence is truncated at the N-terminus by eight residues and there is a serine-for-glycine substitution in the first position of the truncated protein. Both analogues, which were characterized by isoelectric focusing (i.e.f.), polyacrylamide-gel electrophoresis in the presence of SDS (SDS/PAGE), amino acid analysis and N-terminal amino acid sequence determination using combined g.l.c.-m.s., are compared with BGH isolated from pituitaries. In contrast with pituitary-derived BGH, the recombinant-derived proteins are homogeneous on SDS/PAGE and on i.e.f. In a radioimmunoassay, a radioreceptor assay and a bioassay in vivo (rat tibia), Delta-9 BGH showed very similar characteristics to the pituitary-derived hormone. Similar results have also been obtained with the Delta-1 analogue.

SUBMITTER: Wingfield PT

PROVIDER: S-EPMC1147932 | biostudies-other | 1987 May

REPOSITORIES: biostudies-other

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Project description:Background Salmonella is a common foodborne pathogen, which can cause intestinal diseases. In the last decades, the overuse of antibiotics has led to a pandemic of drug-resistant bacterial infections. To tackle the burden of antimicrobial resistant pathogens, it is necessary to develop new antimicrobial drugs with novel modes of action. However, the research and development of antibiotics has encountered bottlenecks, scientific hurdles in the development process, as well as safety and cost challenges. Phages and phage endolysins are promising antibacterial agents that can be used as an alternative to antibiotics. In this context, the expression of endolysin derived from different phages through microbial cells as a chassis seems to be an attractive strategy.Results In this study, a new endolysin from the Salmonella phage XFII-1, named XFII, was screened and obtained. The endolysin yield exceeded 100 mg/mL by heterologous expression from E. coli BL21 and short induction. The endolysin XFII exhibited high bactericidal activity at a concentration of 0.5 μg/mL and reduced the OD600 nm of EDTA-pretreated E. coli JM109 from 0.8 to 0.2 within 5 min. XFII exhibited good thermo-resistance, as it was very stable at different temperatures from 20 to 80℃. Its bactericidal activity could keep constant at 4 °C for 175 days. In addition, the endolysin was able to exert lytic activity in eutrophic conditions, including LB medium and rabbit serum, and the lytic activity was even increased by 13.8% in 10% serum matrices. XFII also showed bactericidal activity against many Gram-negative bacteria, including Salmonella, E. coli, Acinetobacter baumannii, and Klebsiella pneumoniae. Surprisingly, the combination of endolysin XFII and chitosan showed a strong synergy in lysing E. coli and Salmonella without EDTA-pretreatment, and the OD600 nm of E. coli decreased from 0.88 to 0.58 within 10 min.ConclusionsThe novel globular endolysin XFII was screened and successfully expressed in E. coli BL21. Endolysin XFII exhibits a broad lysis spectrum, a rapid and strong bactericidal activity, good stability at high temperatures and under eutrophic conditions. Combined with chitosan, XFII could spontaneously lyse Gram-negative bacteria without pretreatment. This work presented the first characterization of combining endolysin and chitosan in spontaneously lysing Gram-negative bacteria in vitro.

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Preparation and characterization of bovine growth hormones produced in recombinant Escherichia coli.

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