Correlation Of Cell Lysis With High Temperature Stress In The Fermentation Process Of E.coli Expressed ScFv

Cell lysis has been considered as a major process-related issue for a long time while using bacterial host for recombinant protein expression. In this study, an established bioprocess(30oC, p H 7.0, DO 30%) at 5 L bioreactor, where humanized single antibody fragments(Sc Fv) was expressed by Escherichia coli W3110, was used as a model to study the occurrence, causes, molecular mechanism, and process of cell lsyis when Sc Fv expression or the occurrence of high temprature stress(42oC,1.5 h) before or after induction. Meanwhile, the effect of these predetermined stresses on the number of VBNC(Viable but nonculturable cell), the m RNA levels of genes in stresses response pathways and the production of Sc Fv has been investigated. Main results were described as follows:(1) The results of two-level factorial design and analyses of variance indicated that the effect of temperature and the interaction influence between temperature and other factors on the degree of cell lysis was extremely significant. At shake flask level, culture temperature and the interaction influence between temperature and other factors, as well as initial p H showed negative correlation with the degre of cell lysis, while the IPTG concentration, rotation speed s and the interaction influence between these two factors was positively correlated. These findings provided evidence for the latter study about the association of high temprature stress and cell lysis.(2) It was found that the recombinant culture entered viable but nonculturable(VBNC) state at about 6 h earlier than that of wild-type culture, due to the most expressed Sc Fv accumulated within the cell, leading to an accelerated occurrence of cell ysis. Through the transcription level analysis, the process of Sc Fv expression caused the increased expression levels of multiple stressed responses pathway genes, including rpo H, dna K, dna J, gro ES, gro EL of heat shock response pathway, rpo S, gad E, gad X of acid shock response pathway, and sod A, kat E of oxidative stress response pathway. During cell lysis process of recombinant E. coli culture, the expression of membrane protein gene omp A, omp C, omp W and omp X was significantly decreased, and meanwhile rpo E was not sustained at high expression level.(3) The high-temperature stresses before or after induction result that cell lysis occurred 6 h and 1.5 h in advance, respectively, than the control groupat normal culture condition. The results suggested that VBNC state was rapidly induced under the dual stresses of hightemperature stress and Sc Fv expression and the high-temperature stress sooner, the degree of cell lysis deeper. Meanwhile, the high-temperature stress after induction caused the specific production rate declined in advance and the highest concentration of souble Sc Fv was only 59.3% of that in control group. The effect of high-temperature stress before induction on the latter ability of stress response, is greater than high-temperature stress after induction, outer membrane proteins substantially reduce when the process of cell lysis.