Influence of strain and medium composition on filtration of escherichia coli suspensions

Cross-flow filtration of Escherichia coli strains was examined at the laboratory and pilot scales using Romicon 500,000 molecular-weight-cutoff hollow fiber membranes. Both the series resistance and macrosolute polarization models were employed to compare performances. Total dissolved solids content above 90 g/L and viscosity above 1.1 x 10(-3) pac s of cell-free culture media were found to decrease average filtration fluxes by over 60% both in the absence and presence of cells. Broth filtration with culture media of dissolved solids levels below 80 g/L were influenced to a greater extent by harvest cell density. The collodial nature of the complex nutrient responsible for the total solids increase affected prediction of filtration performance. Differences in strain filterability were observed with JM109 preferred over DH5 in high solids-containing media and RR1 preferred over JM109 in low dissolved solids-containing media. Their research demonstrates the importance of cell strain and media selection in the performance of early downstream processing steps. (c) 1994 John Wiley & Sons, Inc.     

TRAIL包涵体在细胞内重折叠现象的初步研究

肿瘤坏死因子相关凋亡诱导配体（Ap02L／TRAIL）是一种新型的抗肿瘤蛋白。但在大肠杆菌表达系统中,表达的TRAIL蛋白往往容易聚集形成包涵体。本文对重组TRAIL包涵体在胞内重折叠转化成可溶性TRAIL蛋白作了初步探索,结果表明：当蛋白表达后,迅速降低温度至30℃并添加50μg／mL氯霉素继续培养4h,可溶性TRAIL产量可提高至16．7％。这一结果在3．7L罐上也得到了证实,可溶性TRAIL表达量达到14．5％。     

A novel technique for the measurement of disruption in high-pressure homogenization: Studies on E. coli containing recombinant inclusion bodies

The high-pressure homogenization of Escherichia coli, strain JM101, containing inclusion bodies of recombinant porcine somatotropin was investigated. A novel technique employing an analytical disc centrifuge was used to monitor the disruption. This a direct technique which measures cell disintegration rather than soluble protein release. The technique is particularly suited to measurements where the disruption approaches 100%. The disk centrifuge provides a size distribution of the homogenate, and furnishes evidence for the preferential disruption of larger cells. For E. coli containing inclusion bodies, and increase in the cell feed concentration from 145 g/L (wet weight) to 330 g/L resulted is poorer homogenization. Poorer disruption was also obtained by lowering the feed temperature from 20 degrees C to 5 degrees C. Only slight variations in performance were obtained by increasing the feed pH from 7.5 to 9.0 or by storing the feed at 4 degrees C for 24 h prior to disruption. Comparison with uninduced E. coli strain JM101, showed that the disruption obtained is higher for bacteria containing a recombinant inclusion body.     

粪便中大肠埃希菌分离方法的筛选

比较了滤膜法、涂布法和纸片法对粪便中大肠埃希菌的分离效果。通过对分离粪便大肠埃希菌的数量可知,纸片法与m—TEC培养基上滤膜法分离的大肠埃希菌数量结果基本一致。m—TEC培养基滤膜法分离的大肠埃希菌平均数量分别是伊红美蓝培养基涂布法分离大肠埃希菌平均数量的1．4倍、伊红美蓝培养基滤膜法分离大肠埃希菌平均数量的2．8倍、m—TEC培养基涂布法分离大肠埃希菌平均数量的2．25倍。分离粪便样品中大肠埃希菌选择滤膜法用m—TEC培齐基进行分离为最佳分离方法。     

The kinetics of RCC1 inclusion body formation in Escherichia Coli

The Regulator of Chromosome Condensation protein (RCC1) is located in both the soluble and inclusion body (IB) fractions of the whole cell lysate when expressed in Escherichia coli BL21 (pLysS) at temperatures below 30 degrees C. When bacterial growth was carried out at 20 degrees C, the majority of the RCC1 remained soluble up to 5.5 h postinduction, When the temperature was raised to 25 degrees C, RCC1 IB was dominant by 1.5 h postinduction. The shift in RCC1 IB formation with temperature suggests that in addition to increased translation rates, folding and aggregation processes may contribute to RCC1 IB formation at higher temperatures. (c) 1995 John Wiley & Sons, Inc.     

Suppression of the Temperature-Sensitive Mutation of the bamD Gene Required for the Assembly of Outer Membrane Proteins by Multicopy of the yiaD Gene in Escherichia coli

We isolated temperature-sensitive mutants of the Escherichia coli bamD gene, which is essential for the assembly of β-barrel outer membrane proteins. As their multicopy suppressor, we identified a novel yiaD gene encoding a putative lipoprotein, YiaD. Mutations of its OmpA domain, which is required for interaction with peptidoglycan, affected suppression, suggesting that interaction with peptidoglycan is important to YiaD function.     

大肠杆菌周质蛋白提取工艺的改进

介绍一种简捷高效的大肠杆菌周质蛋白提取工艺,即用含一定浓度溶菌酶的细胞裂解缓冲液一步提取周质蛋白,与传统的高渗和低渗两步提取法相比,不仅操作简单快捷,并且显著的提高了大肠杆菌周质蛋白的提取率.     

Reassessment of inclusion body-based production as a versatile opportunity for difficult-to-express recombinant proteins

The production of recombinant proteins in the microbial host Escherichia coli often results in the formation of cytoplasmic protein inclusion bodies (IBs). Proteins forming IBs are often branded as difficult-to-express, neglecting that IBs can be an opportunity for their production. IBs are resistant to proteolytic degradation and contain up to 90% pure recombinant protein, which does not interfere with the host metabolism. This is especially advantageous for host-toxic proteins like antimicrobial peptides (AMPs). IBs can be easily isolated by cell disruption followed by filtration and/or centrifugation, but conventional techniques for the recovery of soluble proteins from IBs are laborious. New approaches therefore simplify protein recovery by optimizing the production process conditions, and often include mild resolubilization methods that either increase the yield after refolding or avoid the necessity of refolding all together. For the AMP production, the IB-based approach is ideal, because these peptides often have simple structures and are easy to refold. The intentional IB production of almost every protein can be achieved by fusing recombinant proteins to pull-down tags. This review discusses the techniques available for IB-based protein production before considering technical approaches for the isolation of IBs from E. coli lysates followed by efficient protein resolubilization which ideally omits further refolding. The techniques are evaluated in terms of their suitability for the process-scale production and downstream processing of recombinant proteins and are discussed for AMP production as an example.     

Inactivation of the vascular permeability-increasing activity of bradykinin by mycoplasmas

Mycoplasma pneumoniae, M. genitalium, M. fermentans, M. hominis, M. salivarium, M. orale, Ureaplasma urealyticum and Acholeplasma laidlawii inactivated the vascular permeability-increasing activity of bradykinin when the mixture of bradykinin and mycoplasma cells was injected after incubation at 37 degrees C for 1 h. Cell components responsible for inactivation of the activity of bradykinin were found to be arginine-specific aminopeptidase and carboxypeptidase.     

The outer membrane protein of enteropathogenic Escherichia coli, described as the ''localised adherence factor'', is OmpF and probably not involved in adhesion to HEp-2 cells

Strains of enteropathogenic Escherichia coli (EPEC) were examined for a factor, described as an outer membrane protein (OMP) of 32 kilodaltons (kDa) and reported to be involved in the adhesion of EPEC to HeLa cells. A comparable OMP of 35 kDa was detected in strains of EPEC, although expression of this protein was not related to the ability of strains to adhere to HEp-2 cells. The 35 kDa OMP was found to be heat-modifiable and peptidoglycan associated, and considered to be the porin protein OmpF.