Optimizing the expression of a monoclonal antibody fragment under the transcriptional control of the Escherichia coli lac promoter

The expression of a monoclonal antibody Fab fragment in Escherichia coli strain RB791/pComb3, induced with either lactose or isopropyl-beta-D-thiogalactoside (IPTG), was compared to determine if lactose might provide an inexpensive alternative to induction with IPTG. Induction of Fab expression imposed a metabolic load on the recombinant cells, resulting in lower final cell yields compared to the non-induced controls. An IPTG concentration of 0.05 mM was sufficient to achieve maximal expression of soluble Fab protein when inducing in the early-, mid-, or late-log phases of batch cultures grown using either glucose or glycerol as a carbon source. The largest overall yield of Fab fragments when using 0.05 mM IPTG was achieved by increasing the final yield of cells through glycerol feeding following induction in late-log phase. Lactose was as effective as IPTG for inducing Fab expression in E. coli RB791/pComb3. The greatest overall level of Fab expression was found when cells grown on glycerol were induced with 2 g/L lactose in late-log phase. Since the cost of 0.05 mM of IPTG is significantly greater than the cost of 2 g/L lactose, lactose provides an inexpensive alternative to IPTG for inducing the expression of Fab fragments, and possibly other recombinant proteins, from the E. coli lac promoter.     

IPTG can replace lactose in auto‐induction media to enhance protein expression in batch‐cultured Escherichia coli

Auto‐induction media containing glucose, lactose, and glycerol are a simple and efficient approach for high‐throughput protein expression in Escherichia coli with lac‐derived expression systems. Its principle is based on inducer exclusion between glucose and lactose, preventing the induction by lactose before the depletion of glucose. Isopropyl‐β‐d ‐1‐thiogalactopyranoside (IPTG)—at least in typically used millimolar concentrations—is thought to be unsuitable for this purpose since it can enter the cell by diffusion independently of inducer exclusion. In this study, using parallel batch cultivations in stirred‐tank bioreactors on a milliliter scale, we show that the induction by micromolar concentrations of IPTG is prevented in the presence of glucose. With up to 40 μM IPTG, full induction and heterologous protein expression start only after the depletion of glucose. Thus, auto‐induction is possible with either lactose or IPTG, and the expression greatly depends on the type and concentration of the inducer. The best expression of enhanced green fluorescent protein was achieved with 40 μM IPTG in stirred‐tank bioreactors on a milliliter scale. The IPTG‐based auto‐induction was also reproduced in shaking flasks. Therefore, IPTG can be used in auto‐induction media for protein expression in batch‐cultured E. coli. Furthermore, we show that acetate or arabinose can have significant effects on the auto‐induction mechanism.     

In vivo increase of solubility of overexpressed Hha protein by tandem expression with interacting protein H-NS

Gene cloning in appropriate vectors followed by protein overexpression in Escherichia coli is the common means for protein purification. This approach has many advantages but also some drawbacks; one of these is that many proteins fail to achieve a soluble conformation when overexpressed in E. coli. Hha protein belongs to a family of nucleoid-associated proteins functionally related to the H-NS family of proteins. Hha-like proteins and H-NS-like proteins are able to semidirectly bind to each other. We show in this work that overexpressed Hha or HisHha protein (a functional derivative of Hha containing a 6x His tag at the amino end) from a T7-polymerase promoter in BL21 DE3 E. coli strains results in the vast majority of the protein accumulated in insoluble aggregates (inclusion bodies). We also show that tandem overexpression of HisHha and H-NS increases the solubility of HisHha and prevents the formation of inclusion bodies. Single amino acid substitutions in the HisHha protein, which impair interaction with H-NS, render insoluble protein even when tandem-expressed with H-NS, tandem expression of an insoluble protein and an interacting partner is an experimental strategy which could be useful to increase the solubility of other overexpressed proteins in E. coli.     

A型产气荚膜梭菌α毒素基因表达及其免疫保护作用的初步研究

利用PCR技术,从A型产气荚膜梭菌标准株染色体DNA中扩增出α毒素基因,构建了含α毒素基因的重组菌株BL21(DE3)(pXETA02)。经酶切鉴定和序列测定证实,构建的表达质粒pXETA02含有α毒素基因序列。经SDS-PAGE、Western blot分析和ELISA检测,重组菌株表达的α毒素蛋白能够被α毒素单抗识别。表达优化结果表明,以IPTG为诱导剂诱导α毒素表达的优化条件是:培养基pH 7.5,培养温度37℃,IPTG浓度0.8mmol/L,菌体生长密度OD600达到0.8时加入IPTG,诱导时间5h,此时α毒素蛋白表达量为34.83%。以乳糖为诱导剂诱导α毒素表达的优化条件是:培养基pH7.5、培养温度37℃,乳糖浓度0.1g/L,菌体生长密度OD600达到0.8时加入乳糖,诱导时间5h,α毒素蛋白表达量为23.82%。动物实验结果表明,用重组菌株α毒素蛋白免疫的小鼠可以抵抗1MLD的A型产气荚膜梭菌标准株C57-1毒素攻击。     

Lactose Induction Increases Production of Recombinant Keratinocyte Growth Factor-2 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Recombinant human keratinocyte growth factor-2 (rhKGF-2) has previously been expressed in Escherichia coli using isopropyl-β-d-thiogalactopyranoside (IPTG), a non-metabolizable and expensive compound, as the inducer. In order to determine whether IPTG could be replaced with the cheap and natural lactose to induce rhKGF-2 expression, we examined the expression of rhKGF-2 in flask culture and 30-l fermentation using lactose as the inducer. The optimized fermentation induced with lactose resulted in 1,382 g of cell mass, corresponding to a 84% enhancement in cell mass compared with IPTG induction. While the expression level of rhKGF-2 induced with lactose was comparable to that induced with IPTG, the solubility of target protein was increased by lactose induction than by IPTG induction. The recombinant protein was further purified by cation exchange and heparin-affinity chromatography. 255 milligrams of pure rhKGF-2 was achieved per liter culture by lactose induction, 52% higher than that obtained by IPTG induction. A preliminary biochemical characterization of purified rhKGF-2 was performed by Western blotting and mitogenic activity analysis, and the results demonstrated that the purified lactose-induced rhKGF-2 could react with anti-human KGF-2 antibody and stimulate the proliferation of FGFR2-IIIb-transfected mouse BaF3 cells as IPTG-induced rhKGF-2 could do.     

Effect of growth temperature,induction, and molecular chaperones on the solubilization of over-expressed cellobiose phosphorylase from <Emphasis Type="Italic">Cellvibrio Gilvus</Emphasis> under <Emphasis Type="Italic">in vivo</Emphasis> conditions

In vivo folding of many proteins can be facilitated by growth temperature, extent of induction, and molecular chaperones, which prevent over-expressed protein from being trapped into insoluble inclusion bodies. In the present report, we describe the role of molecular chaperones and growth temperature on the solubilization of overexpressed Cellobiose Phosphorylase (CBP) in Escherichia coli. The growth of host at low temperature enhanced enzyme in soluble fraction. Similarly, induction of target gene at low level of IPTG also yielded higher enzyme in soluble fraction. The synergistic effect of low temperature and induction on the prevention of inclusion bodies was also evident from our results. In addition, co-expression of the target gene with two types of molecular chaperones (GroESL and KODHsp) was also attempted. However, none of these chaperones enhanced the solubilization under in vivo conditions. Nevertheless, effective role of low growth temperature coupled with low level of induction appeared to be an attractive feature for producing recombinant protein.     

乳糖作为诱导剂对重组目的蛋白表达的影响

将重组粒细胞-巨噬细胞集落刺激因子/白细胞介素3(GM-CSF/IL-3)融合蛋白表达菌BL21(DE3)(pFu)作为研究对象,对于以乳糖作为诱导剂时重组目的产物的诱导表达规律进行了深入的研究。分析比较了不同培养基中,不同生长阶段进行诱导对于产物表达的影响。对诱导所需的乳糖浓度、诱导持续时间长短等因素亦进行了研究。实验结果表明,在对诱导条件进行优化控制的前提下,利用乳糖作为诱导剂可以达到与IPTG类似的诱导效果。随后的研究中,将乳糖作为诱导剂应用于高密度发酵过程。这些研究结果为乳糖作为诱导剂最终应用于重组基因工程药物的工业化生产提供了有益的参考和借鉴。     

乳糖代替IPTG诱导己糖激酶在大肠杆菌中的表达

构建了己糖激酶GLK高效表达的工程菌株BL21(DE3)/pET-glk,考察了乳糖代替IPTC诱导己糖激酶GLK在大肠杆菌BL21(DE3)中表达的可行性.实验结果表明,工程菌对数生长中期(OD<,600>约为1.0)添加终浓度为10 g/L的乳糖于25℃的条件下诱导6 h能获得最大量的目的蛋白和菌体量,目的蛋白表达...     

Characterization of SotA and SotB, two Erwinia chrysanthemi proteins which modify isopropyl-beta-D-thiogalactopyranoside and lactose induction of the Escherichia coli lac promoter

The expression, in Escherichia coli, of variants of the Erwinia chrysanthemi secretion genes outB and outS under the Ptac promoter is toxic to the cells. During attempts to clone E. chrysanthemi genes able to suppress this toxicity, I identified two genes, sotA and sotB, whose products are able to reduce the isopropyl-beta-D-thiogalactopyranoside (IPTG) induction of the E. coli lac promoter. SotA and SotB belong to two different families of the major facilitator superfamily. SotA is a member of the sugar efflux transporter family, while SotB belongs to the multidrug efflux family. The results presented here suggest that SotA and SotB are sugar efflux pumps. SotA reduces the intracellular concentration of IPTG, lactose, and arabinose. SotB reduces the concentration of IPTG, lactose, and melibiose. Expression of sotA and sotB is not regulated by their substrates, but sotA is activated by the cyclic AMP receptor protein (CRP), while sotB is repressed by CRP. Lactose is weakly toxic for E. chrysanthemi. This toxicity is increased in a sotB mutant which cannot efficiently efflux lactose. This first evidence for a physiological role of sugar efflux proteins suggests that their function could be to reduce the intracellular concentration of toxic sugars or sugar metabolites.     

乳糖诱导木聚糖酶基因在大肠杆菌中的可溶性表达

以构建好的大肠杆菌工程菌BL21(DE3)/xylanase为研究对象,研究了以IPTG和乳糖作为诱导剂时重组蛋白的表达规律。在摇瓶发酵条件下研究了诱导剂浓度、诱导时机、诱导培养时间和诱导培养温度对目标蛋白表达的影响。实验结果表明,乳糖作为诱导剂时,重组菌产酶活力33.9 U/mg略高于IPTG作为诱导剂时重组菌产酶活力28.10 U/mg,这为乳糖作为诱导剂应用于重组大肠杆菌生产木聚糖酶提供了参考依据。     

Protein production by auto-induction in high density shaking cultures

Inducible expression systems in which T7 RNA polymerase transcribes coding sequences cloned under control of a T7lac promoter efficiently produce a wide variety of proteins in Escherichia coli. Investigation of factors that affect stability, growth, and induction of T7 expression strains in shaking vessels led to the recognition that sporadic, unintended induction of expression in complex media, previously reported by others, is almost certainly caused by small amounts of lactose. Glucose prevents induction by lactose by well-studied mechanisms. Amino acids also inhibit induction by lactose during log-phase growth, and high rates of aeration inhibit induction at low lactose concentrations. These observations, and metabolic balancing of pH, allowed development of reliable non-inducing and auto-inducing media in which batch cultures grow to high densities. Expression strains grown to saturation in non-inducing media retain plasmid and remain fully viable for weeks in the refrigerator, making it easy to prepare many freezer stocks in parallel and use working stocks for an extended period. Auto-induction allows efficient screening of many clones in parallel for expression and solubility, as cultures have only to be inoculated and grown to saturation, and yields of target protein are typically several-fold higher than obtained by conventional IPTG induction. Auto-inducing media have been developed for labeling proteins with selenomethionine, 15N or 13C, and for production of target proteins by arabinose induction of T7 RNA polymerase from the pBAD promoter in BL21-AI. Selenomethionine labeling was equally efficient in the commonly used methionine auxotroph B834(DE3) (found to be metE) or the prototroph BL21(DE3).     

Increasing the yield of soluble recombinant protein expressed in E. coli by induction during late log phase

Recombinant mammalian proteins expressed in E. coli can be difficult to purify in high yield in a soluble and functional form. Various techniques have been described to prevent proteolysis of expressed proteins and/or their sequestering as insoluble aggregates within inclusion bodies. We report conditions for expressing recombinant proteins from E. coli that significantly enhanced the yield of soluble and functional protein. We demonstrate high-yield recovery of a native, high-molecular-weight RNA binding protein without the aid of fusion protein sequence. The principle factor that increased protein yield was the induction of protein expression in a late log phase culture, although reduced temperature during the induction and a low IPTG concentration also contributed to a higher yield.     

乳糖诱导的重组人血管内皮抑素(rhEndostatin)蛋白的表达

研究用乳糖替代IPTG作为诱导剂进行重组蛋白的表达,观察乳糖对乳糖操纵子调控的基因工程菌发酵及重组血管内皮抑素表达的影响,从而选取最佳诱导表达条件。以重组人血管内皮抑素表达工程菌pETrhEN/BL21(DE3)作为研究对象,分别用IPTG和乳糖作为诱导剂,在摇瓶中进行表达实验。并对重组蛋白质表达量进行分析。然后在5 L发酵罐中进行验证。在摇瓶培养条件下,乳糖浓度大于0.5 g/L即可以诱导目的蛋白的表达。乳糖浓度1 g/L时诱导目的蛋白表达量与1 mmol/L的IPTG相当,当乳糖浓度为10 g/L,目的蛋白表达量达到最大。在发酵罐培养条件下,补料4 h后葡萄糖浓度基本耗尽,此时开始加入乳糖。诱导后1 h,即有重组蛋白表达,在诱导后4 h达到高峰(占菌体可溶性蛋白的56%),与此同时,诱导后5 h菌体浓度也达到最高值。在以乳糖操纵子为调控手段的工程菌表达系统中,可以使用乳糖作为诱导剂,诱导应在葡萄糖消耗完后进行。     

Functional analysis of the GAF domain of NifA in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>: effects of Tyr→Phe mutations on NifA and its interaction with GlnB

Regulation of NifA activity in Azospirillum brasilense depends on GlnB (a PII protein), and it was previously reported that the target of GlnB activity is the N-terminal domain of NifA. Furthermore, mutation of the Tyr residue at position 18 in the N-terminal domain resulted in a NifA protein that did not require GlnB for activity under nitrogen fixation conditions. We report here that a NifA double mutant in which the Tyr residues at positions 18 and 53 of NifA N-were simultaneously replaced by Phe (NifA-Y1853F) displays high nitrogenase activity, which is still regulatable by ammonia, but not by GlnB. The yeast two-hybrid technique was used to investigate whether GlnB can physically interact with wild-type and mutant NifA proteins. GlnB was found to interact directly with the N-terminal GAF domain of wild-type NifA, but not with its central or C-terminal domain. GlnB could still bind to the single NifA mutants Y18F and Y53F. In contrast, no interaction was detected between GlnB and the double mutant NifA-Y18/53F or between GlnB and NifA-Y43.     

High yield expression of recombinant pro-resilin: lactose-induced fermentation in E. coli and facile purification

Resilin is an elastic protein with outstanding material properties: high resilience and a very high fatigue lifetime. We are interested in the production of resilin-like proteins which can be photo-chemically cross-linked to form rubbery biomaterials to be used in a variety of industrial and medicinal applications. A method has been developed for producing soluble recombinant proteins in small scale fermentation equipment using glycerol batch for initial growth and primary induction by IPTG at carbon source depletion, followed by new growth in lactose-induced culture. Recombinant rec1-resilin has been over-expressed in the host strain Escherichia coli BL21(DE3)pLysS at a level of up to 300 mg/l, a greater than 20-fold increase in volumetric productivity, relative to that obtained from conventional IPTG induction in LB medium. The primary induction step before lactose induction in fresh medium resulted in a 2.5- to 3-fold increase of both volumetric productivity and cell specific yield compared to that without primary induction under the same conditions. This method is amenable and suitable for large scale production of soluble resilin-like proteins at a low operating cost. In addition, a simple 'salt precipitation and heat purification' method allowed rapid and efficient downstream processing of a large quantity of soluble recombinant resilin-like proteins. These methods will enable investigation of the structural and functional properties of resilin-like proteins, and the development of highly resilient biomaterials.     

argE基因在大肠杆菌中的表达优化

N-乙酰鸟氨酸脱酰基酶可在重组菌BL21（DE3）-pET22b-argE中表达。首先确定了该酶的细胞表达定位,再研究了诱导温度、诱导剂种类及浓度、诱导起始菌体密度、诱导时间等因素对重组菌生长及目的蛋白表达活性的影响。结果表明,IPTG和乳糖皆可诱导目的蛋白表达,乳糖的诱导效果优于IPTG。在诱导起始0D600为0．46时加入15g／L乳糖,20℃诱导18h最适于目的蛋白的活性表达。表达条件优化后,酶活从1．68U／mL提高至282．99U／mL,约为原来的168倍。