James F. Kane

Formation of recombinant protein inclusion bodies in Escherichia coli

Overexpression of cloned genes in Escherichia coli may lead to the formation of intracellular proteinaceous granules that are readily visible under the light microscope. A number of parameters relating to the host cell, the growth conditions and the properties of the particular protein affect this process. This short review considers some of these conditions and proposes a model to explain the formation of the refractible particles.

Fate of Recombinant Escherichia coli K-12 Strains in the Environment

Publisher Summary This chapter discusses the fate of recombinant Escherichia coli K-12 strains in the environment. The strains of E. coli K-12 are unable to adhere to mammalian intestinal cells and do not colonize the conventional mammalian intestinal tract. The E. coli K-12 strains are able to colonize germ-free or antibiotic-treated rats and mice but not antibiotic-treated humans. The presence of conjugative and nonconjugative plasmids was observed to impart an additional disadvantage to E. coli K-12 strains in these environments, particularly in the mammalian intestinal tract. For the conjugational transfer of pBR322 or a derivative of pBR322, several conditions must be met. The pBR322 plasmid must have intact bom and nic sites; a derivative, such as pBR327, which lacks these two sites, is unable to be transferred. The fate of recombinant E. coli K-12 strains in the environment is an important and interesting issue that can continue to attract increased attention.

Fate in sewage of a recombinantEscherichia coli K-12 strain used in the commercial production of bovine somatotropin

SummaryThe fate of a derivative ofEscherichia coli strain W3110G [pBGH1], a strain used for production of bovine somatotropin, was examined in semi-continuous activated sludge (SCAS) units. A nalidixic acid-resistant derivative of W3110G [pBGH1], strain LBB270 [pBGH1], was used to facilitate tracking. SCAS units (300 ml) containing municipal mixed liquor were operated on a daily cycle of 23 h aeration and 1 h settling followed by decanting of clear supernatant (175 ml) and refilling with fresh primary effluent. SCAS units were inoculated with two concentrations ofE. coli LBB270 [pBGH1] and operated for 200 h. Viable levels ofE. coli LBB270 [pBGH1] were measured daily in aerated mixed liquor and decanted supernatant. Viable counts in the mixed liquor decreased from 10000- to 100000-fold in less than 200 h. Losses ofE. coli LBB270 [pBGH1] in decanted supernatants accounted for less than 2-fold of the total losses observed in the SCAS units. TheE. coli LBB270 [pBGH1] was not evenly distributed in the mixed liquor, but became preferentially associated with the settleable floc. These results show thatE. coli LBB270 [pBGH1] was unable to survive in municipal sludge even when inoculated at concentrations greater than, or comparable to, levels of indigenous microorganisms.

Environmental assessment of recombinant DNA fermentations

SummaryThis issue of theJournal of Industrial Microbiology contains a compilation of papers presented at the 1992 National Meeting of the Society for Industrial Microbiology in two symposia entitled ‘Environmental Assessment of Recombinant DNA Fermentations’. It focuses on three areas of particular interest to industry usingEscherichia coli K-12 strains to make recombinant proteins: (i) the current regulatory environment; (ii) plant design; (iii) results from five different companies all of whom are using or planning to use recombinantE. coli in commercial fermentations. The results from all five companies pursuing the questions of environmental fate and the potential for gene transfer in different studies reached the same conclusions. That is, recombinantE. coli K-12 strains and their plasmidless hosts were unable to survive in any environmental microcosm tested. Additionally, there was absolutely no evidence of gene transfer despite the use of highly sensitive techniques to measure such an event. It seems reasonable to conclude thatE. coli K-12 strains with recombinant, non-conjugating, poorly mobilizable plasmids do not represent environmental hazards in the event of an accidental release of such microorganisms into the environment.

Potential for gene transfer from recombinantEscherichia coli K-12 used in bovine somatotropin production to indigenous bacteria in river water

SummaryThis study examined the transfer of the plasmid pBGH1, an expression vector for bovine somatotropin (BST), fromEscherichia coli K-12 strain W3110G [pBGH1] to indigenous microorganisms present in flasks containing Missouri River water. Strain LBB269 is a nalidixic acid-resistant derivative of W3110G which was used as a plasmid-free control strain in these studies. Water samples were inoculated with strains W3110G [pBGH1] and LBB269; after 21 days of incubation the number of viable colony-forming units (CFU) of W3110G [pBGH1] and LBB269 were reduced from an initial level of about 1×107 CFU per ml to less than 1 CFU per 100 ml. At this time indigenous microbes resistant to both ampicillin and tetracycline (the antibiotic resistance markers on pBGH1) were isolated from 100 ml of water from each of the flasks inoculated with either strain W3110G [pBGH1] or LBB269. Plasmid DNA was isolated from these organisms and examined for sequences containing the gene for BST from pBGH1, using a polymerase chain reaction (PCR) assay. As expected, the day 0 sample from the flask inoculated withE. coli K-12 strain W3110G [pBGH1] gave a positive PCR response and the day 0 sample from the flask inoculated withE. coli K-12 strain LBB269 gave a negative PCR response. All of the day 21 samples containing indigenous microbes isolated from flasks that were inoculated with either W3110G [pBGH1] or LBB269 were negative in the PCR assay, indicating that the target sequence from pBGH1 was not present in any of these indigenous microorganisms. The results of this particular assay indicate that pBGH1 or the portion of pBGH1 including the BST structural gene had not been transferred from W3110G [pBGH1] to indigenous microbial inhabitants of the Missouri River water flasks during this study.

Isolation of Escherichia coli synthesized recombinant proteins that contain ε-N-acetyllysine

Publisher Summary This chapter describes the presence of significant amounts of ɛ-N-acetyllysine in rpST and rbST, eukaryotic proteins expressed in a prokaryotic system. ESMS, amino acid sequencing and amino acid analyses demonstrate the presence of ɛ-N-acetyllysine in two recombinant proteins; bovine placental lactogen and human tissue factor pathway inhibitor. These data establish that this modified amino acid is present in several distinct recombinant eukaryotic proteins expressed in E.coli . Formation of ɛ-N-acetyllysine in eukaryotic systems involves a post-translational mechanism in which the acetyltransferase uses acetyl-CoA as the source of the acetyl group. It may be possible that acetylation of lysines occurs by a chemical mechanism with acetyl-CoA or some other metabolic intermediate providing the source of the acetyl group. The chapter explains that acetylation of lysines is an important modification, which can occur during the expression of recombinant proteins expressed in E.coli. However, investigations into the effects of fermentation conditions on the level of ɛ-N-acetyllysine formation may lead to a better understanding of this event.