Enhanced production of D-(-)-3-hydroxybutyric acid by recombinant Escherichia coli

Wild-type bacteria including Escherichia coli normally do not produce extracellular D-(-)-3-hydroxybutyric acid (3HB). To produce extracellular chiral 3HB, a new pathway for synthesis of 3HB was constructed by simultaneous expression of genes of beta-ketothiolase (phbA), acetoacetyl-CoA reductase (phbB), phosphor-transbutyrylase (ptb) and butyrate kinase (buk) in E. coli strain DH5alpha. E. coli DH5alpha containing any one of the four plasmids pBHR69, pUCAB, p68CM or pKKAB that harbor the phbA and phbB genes produced small amounts of 3HB, ranging from 75 to 400 mg l(-1), while E. coli DH5alpha harboring p68CMPTK containing genes of phbA, phbB, ptb and buk increased the 3HB concentration to 1.4 g l(-1) in shake flasks supplemented with LB broth and 20 g l(-1) glucose. 3HB production was further improved to over 2 g l(-1) in shake flasks when E. coli DH5alpha hosted two plasmids simultaneously that separately contained phbA and phbB in one plasmid while ptb and buk in the other. A batch fermentation run in a 5-l fermenter produced approximately 5 g l(-1) 3HB after 24 h. A fed-batch process increased 3HB production to 12 g l(-1) after 48 h of fermentation.     

Optimization of the AT-content of Codons Immediately Downstream of the Initiation Codon and Evaluation of Culture Conditions for High-level Expression of Recombinant Human G-CSF in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Enhanced therapeutic importance of recombinant human granulocyte colony stimulating factor (rhG-CSF) has encouraged us to develop a processing method for its high-level expression in E. coli. In this study, we established a high-yielding clone by incorporation of silent mutations at N-terminal region of human G-CSF gene. We studied and optimized various parameters of culture conditions connected with the expression of rhG-CSF. The maximum expression was obtained in a defined medium supplemented with 1% glucose. The gene in pET-3a vector in E. coli BL21 (DE3) PLysS host strain was induced with 2 mM isopropyl β-d-1-thiogalacto pyronoside. The cell growth and productivity was enhanced about 1.6- and 1.5-folds, respectively when inducing the culture at OD₆₀₀ value of 6 than 2. The protein expression was significantly increased by addition of rifampicin at concentration of 200 μg/ml. The AT content of 51.8% with suitable codon sequences at N-terminal region and the concentration of rifampicin were identified as the key factors with a significant impact on protein expression. The specific productivity of 104 mg/OD/l (68.7% of total cellular protein) of rhG-CSF was obtained toward the end of the study, which is almost 1.5 times higher yield than reported so far in the literature.     

Production of D-(--)-3-hydroxyalkanoic acid by recombinant Escherichia coli

Pathways for extracellular production of chiral D-(-)-3-hydroxybutyric acid (3HB) and D-(-)-3-hydroxyalkanoic acid (mcl-3HA) were constructed by co-expression of genes of beta-ketothiolase (phbA), acetoacetyl-CoA reductase (phbB) and 3-hydroxyacyl-ACP CoA transacylase (phaG), respectively, in Escherichia coli strain DH5alpha. The effect of acrylic acid and glucose on production of both 3HB and mcl-3HA was investigated. It was found that the addition of acrylic acid significantly increased production of 3HB and mcl-3HA consisting of 3-hydroxyoctanoic acid and 3-hydroxydecanoic acid in a ratio of 1:3 from 199 mg x l(-1) to 661 mg x l(-1) and from 27 mg x l(-1) to 135 mg x l(-1), respectively, in shake flask studies when glucose was present in the medium at the very beginning of fermentation. The timing of glucose addition had no effect on 3HB production. In contrast, mcl-3HA production was affected by glucose addition, an mcl-3HA concentration of 193 mg x l(-1) was obtained when glucose was added to the culture at 12 h. A more than seven-fold increase was obtained when compared with that in medium containing glucose at the beginning of fermentation. However, a decrease in production of 3HB and mcl-3HA was found when glucose was added at 12 h to the culture containing acrylic acid. The repressive effect of acrylic acid on acetic acid production was also evaluated and discussed.     

Over-expression of recombinant human interferon-gamma in high cell density fermentation of Escherichia coli

Human interferon-gamma (hIFN-gamma) was expressed in Escherichia coli BL21(DE3) under the control of the T7 promoter. Glucose was used as the sole source of carbon and energy with simple exponential feeding rate in fed-batch process. Cell density of recombinant E. coli was reached to 100 g dry wt l(-1) under both constant (0.12 h(-1)) and variable (0.12-0.52 h(-1)) specific growth rates. In the variable specific growth rate fed-batch process, plasmid stability and specific yield of rhIFN-gamma were greater than constant specific growth rate fed-batch process. The final specific yield and overall productivity of rhIFN-gamma were 0.35 +/- 0.02 g rhIFN-gamma g(-1) dry cell wt and 0.9 +/- 0.05 g rhIFN-gamma l(-1) h(-1) in the variable specific growth rate fed-batch process, respectively.     

Proposed mechanism of acetate accumulation in two recombinant Escherichia coli strains during high density fermentation

The productivity of Escherichia coli as a producer of recombinant proteins is affected by its metabolic properties, especially by acetate production. Two commercially used E. coli strains, BL21 (lambdaDE3) and JM109, differ significantly in their acetate production during batch fermentation at high initial glucose concentrations. E. coli BL21 grows to an optical density (OD, 600 nm) of 100 and produces no more than 2 g/L acetate, while E. coli JM109 grows to an OD (600 nm) of 80 and produces up to 14 g/L acetate. Even in fed-batch fermentation, when glucose concentration is maintained between 0.5 and 1.0 g/L, JM109 accumulates 4 times more acetate than BL21. To investigate the difference between the two strains, metabolites and enzymes involved in carbon utilization and acetate production were analyzed (isocitrate, ATP, phosphoenolpyruvate, pyruvate, isocitrate lyase, and isocitrate dehydrogenase). The results showed that during batch fermentation isocitrate lyase activity and isocitrate concentration were higher in BL21 than in JM109, while pyruvate concentration was higher in JM109. The activation of the glyoxylate shunt pathway at high glucose concentrations is suggested as a possible explanation for the lower acetate accumulation in E. coli BL21. Metabolic flux analysis of the batch cultures supports the activity of the glyoxylate shunt in E. coli BL21.     

Metabolic engineering of Escherichia coli for production of enantiomerically pure (R)-(--)-hydroxycarboxylic acids

A heterologous metabolism of polyhydroxyalkanoate (PHA) biosynthesis and degradation was established in Escherichia coli by introducing the Ralstonia eutropha PHA biosynthesis operon along with the R. eutropha intracellular PHA depolymerase gene. By with this metabolically engineered E. coli, enantiomerically pure (R)-3-hydroxybutyric acid (R3HB) could be efficiently produced from glucose. By employing a two-plasmid system, developed as the PHA biosynthesis operon on a medium-copy-number plasmid and the PHA depolymerase gene on a high-copy-number plasmid, R3HB could be produced with a yield of 49.5% (85.6% of the maximum theoretical yield) from glucose. By integration of the PHA biosynthesis genes into the chromosome of E. coli and by introducing a plasmid containing the PHA depolymerase gene, R3HB could be produced without plasmid instability in the absence of antibiotics. This strategy can be used for the production of various enantiomerically pure (R)-hydroxycarboxylic acids from renewable resources.     

抗菌肽Adenoregulin基因工程菌培养条件的优化及分批发酵研究

denoregulin（ADR）是来源于南美树蛙Phyllomedusa bicolor皮肤的含有33个氨基酸的抗菌肽,在非极性环境中形成α_螺旋型结构,具有抗菌活性强、抗菌谱广的特点。将ADR基因克隆于pET32a载体上,转化大肠杆菌BL21(DE3),对这一工程菌株的培养条件进行了优化。通过正交试验,考察诱导时机、诱导剂量和诱导时间三个因素的不同水平对蛋白表达的影响,结果发现诱导时机的影响尤为显著,考察了9种不同培养基对表达量的影响,发现培养基中加入葡萄糖对目标蛋白的稳定表达起了重要的作用,确定最佳培养条件为：培养基为2×YT+0.5%葡萄糖,诱导时机为OD600=0.9左右,诱导剂IPTG加入的终浓度为0.1mmol/L,诱导时间为4h。采用前期恒pH、后期指数流加的策略进行工程菌BL21(DE3)/pET32a-adr的高密度培养,在整个流加过程中,通过控制葡萄糖的加入量,将菌株的比生长速率控制在015h^-1,乙酸浓度也被控制在较低的水平(<2g/L),但是质粒丢失严重,在发酵结束时,约有40%的大肠杆菌中不带质粒,这导致了目标蛋白的表达量下降严重,但是表达的目标蛋白90%以上为可溶性形式。表达的融合蛋白无抑菌活性,而裂解后得到的ADR单体具有明显的抑菌活性。     

以葡萄糖为唯一碳源合成聚-4-羟基丁酸的重组大肠杆菌的构建

为实现重组大肠杆菌以葡萄糖为唯一碳源合成均聚的P( 4HB) ,PCR扩增大肠杆菌编码谷氨酸:琥珀酰半缩醛转氨基酶基因(gabT) ,谷氨酸脱羧酶基因(gadA)以及富养罗尔斯通氏菌(Ralstoniaeutropha)H16的4_羟基丁酸脱氢酶基因(gadB) ,并组装到携带富养罗尔斯通氏菌(Ralstoniaeutropha)H16的PHA聚合酶基因(phaC)和克氏梭菌(Clostridiumkluyveri)中编码4_羟基丁酸:CoA转移酶基因(orfZ)的重组质粒pKESS5 3上,形成一个大的操纵元。携带重组质粒的大肠杆菌获得从三羧酸循环的中间物———α_酮戊二酸到P( 4HB)的代谢途径。结果表明,重组大肠杆菌可以以葡萄糖为唯一碳源合成均聚的P( 4HB) ,当向以葡萄糖为唯一碳源的无机培养基添加蛋白胨、酵母提取物、酪蛋白水解物时,P( 4HB)的含量可以高达菌体干重的30 %。     

Analysis of in vivo substrate specificity of the PHA synthase from Ralstonia eutropha: formation of novel copolyesters in recombinant Escherichia coli

In order to investigate the in vivo substrate specificity of the type I polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha, we functionally expressed the PHA synthase gene in various Escherichia coli mutants affected in fatty acid beta-oxidation and the wild-type. The PHA synthase gene was expressed either solely (pBHR70) or in addition to the R. eutropha genes encoding beta-ketothiolase and acetoacetyl-coenzyme A (CoA) reductase comprising the entire PHB operon (pBHR68) as well as in combination with the phaC1 gene (pBHR77) from Pseudomonas aeruginosa encoding type II PHA synthase. The fatty acid beta-oxidation route was employed to provide various 3-hydroxyacyl-CoA thioesters, depending on the carbon source, as in vivo substrate for the PHA synthase. In vivo PHA synthase activity was indicated by PHA accumulation and substrate specificity was revealed by analysis of the comonomer composition of the respective polyester. Only in recombinant E. coli fad mutants harboring plasmid pBHR68, the R. eutropha PHA synthase led to accumulation of poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) (poly(3HB-co-3HO)) and poly(3HB-co-3HO-co-3-hydroxydodecanoate (3HDD)), when octanoate and decanoate or dodecanoate were provided as carbon source, respectively. Coexpression of phaC1 from P. aeruginosa indicated and confirmed the provision of PHA precursor via the beta-oxidation pathway and led to the accumulation of a blend of two different PHAs in the respective E. coli strain. These data strongly suggested that R. eutropha PHA synthase accepts, besides the main substrate 3-hydroxybutyryl-CoA, also the CoA thioesters of 3HO and 3HDD.     

Expression of ornithine decarboxylase of Coccidioides immitis in three Escherichia coli strains carrying the lambda DE3 lysogen and an E. coli EWH319 strain odc − null mutant

Ornithine decarboxylase from respiratory fungal pathogen, Coccidioides immitis, cloned in the pETCiODC plasmid under control of T7lac promoter, was produced in E. coli BL21(DE3), BL21(DE3)pLysS, BLR(DE3) and EWH319 transformant strains. E. coli BL21(DE3)pLysS-pETCiODC expressed the highest specific activity of ODC, suggesting that this strain could be successfully used for protein structure and drug testing studies.     

利用Duet系列共表达载体构建工程菌生物合成3-羟基丁酸(3HB)

通过PCR获得β-酮基硫解酶(PhaA)、乙酰乙酰辅酶A还原酶(PhaB)和硫酯酶(TesB)的基因phaA,phaB和tesB。以pACYCDuet-1、pRSFDuet-1、PET30a(+)为载体,以大肠杆菌表达型宿主E.coli BL21(DE3)为宿主茵,分别构建了两株表达体系不同的工程茵,建立了R-3-羟基丁酸(3HB)的代谢途径,实现3HB的生物合成。将两株工程茵分别进行诱导表达及发酵,结果表明,三个基因均可在表达型宿主BL21(DE3)中进行活性表达,通过三个酶的连续催化生物合成3HB,并且三个基因的均衡表达更有利于3HB的生物合成。将phaAB基因簇构建在ACYCDuet-1上,tesB基因构建在RSFDuet-1上,获得的工程茵BL21(AAB+RB),3HB产量可达到1.8 g/L。     

牛流行热病毒G1抗原表位基因在大肠杆菌中的表达、纯化及抗原性鉴定

从包含牛流行热病毒G蛋白基因的质粒pMD-G中克隆G1抗原表位区基因,与表达载体pGEX-4T-1连接,成功构建重组质粒pGEX-G₁。重组质粒转化BL21（DE3）,以IPTG进行诱导,并确定了最佳表达条件的IPTG浓度为0.1mmol/L、反应温度为16℃、诱导时间为18h。可溶性表达的目的蛋白经Glutathione Sepharose TM^4B介质纯化,纯度达80%;以包涵体形式存在的重组蛋白以2%的脱氧胆酸钠洗涤、0.5%的N-十二烷基肌氨酸钠溶解、透析复性、Glutathione Sepharose TM^4B纯化后,纯度达85%以上。Western blot试验表明纯化的目的蛋白有良好的反应原性。经间接ELISA检测,测得牛流行热病毒12份阳性血清的OD490值平均为1.813±0.231,12份阴性血清的OD490值平均为0.359±0.032,差异极显著（P<0.01）。将重组蛋白作为抗原免疫兔子,试验兔均产生了高滴度的抗体,证实该蛋白有免疫原性。将目的蛋白作为包被抗原,测得8份狂犬病病毒阳性血清的OD490值平均为0.324±0.031,与所测12份阴性血清的OD490值接近,说明不存在交叉反应。以上结果均证实纯化后的重组蛋白有良好的生物学活性和特异性,可作为包被抗原,开发ELISA试剂盒。     

Hyperproduction of poly(4-hydroxybutyrate) from glucose by recombinant Escherichia coli

ABSTRACT: BACKGROUND: Poly(4-hydroxybutyrate) [poly(4HB)] is a strong thermoplastic biomaterial with remarkable mechanical properties, biocompatibility and biodegradability. However, it is generally synthesized when 4-hydroxybutyrate (4HB) structurally related substrates such as gamma-butyrolactone, 4-hydroxybutyrate or 1,4-butanediol (1,4-BD) are provided as precursor which are much more expensive than glucose. At present, high production cost is a big obstacle for large scale production of poly(4HB). RESULTS: Recombinant Escherichia coli strain was constructed to achieve hyperproduction of poly(4-hydroxybutyrate) [poly(4HB)] using glucose as a sole carbon source. An engineering pathway was established in E. coli containing genes encoding succinate degradation of Clostridium kluyveri and PHB synthase of Ralstonia eutropha. Native succinate semialdehyde dehydrogenase genes sad and gabD in E. coli were both inactivated to enhance the carbon flux to poly(4HB) biosynthesis. Four PHA binding proteins (PhaP or phasins) including PhaP1, PhaP2, PhaP3 and PhaP4 from R. eutropha were heterologously expressed in the recombinant E. coli, respectively, leading to different levels of improvement in poly(4HB) production. Among them PhaP1 exhibited the highest capability for enhanced polymer synthesis. The recombinant E. coli produced 5.5 g L-1 cell dry weight containing 35.4% poly(4HB) using glucose as a sole carbon source in a 48 h shake flask growth. In a 6-L fermentor study, 11.5 g L-1 cell dry weight containing 68.2% poly(4HB) was obtained after 52 h of cultivation. This was the highest poly(4HB) yield using glucose as a sole carbon source reported so far. Poly(4HB) was structurally confirmed by gas chromatographic (GC) as well as 1H and 13C NMR studies. CONCLUSIONS: Significant level of poly(4HB) biosynthesis from glucose can be achieved in sad and gabD genes deficient strain of E. coli JM109 harboring an engineering pathway encoding succinate degradation genes and PHB synthase gene, together with expression of four PHA binding proteins PhaP or phasins, respectively. Over 68% poly(4HB) was produced in a fed-batch fermentation process, demonstrating the feasibility for enhanced poly(4HB) production using the recombinant strain for future cost effective commercial development.     

Expression, refolding, and characterization of a novel recombinant dual human stem cell factor

A novel recombinant dual human stem cell factor (rdhSCF) gene which consisted of a full-length hSCF(1-165aa) cDNA and a truncated hSCF (1-145aa) cDNA, linked by a peptide (GGGGSGGGGSGG) coding region, was constructed and cloned into Escherichia coli expression vector pET-22b. The rdhSCF was expressed at high level in E. coli BL21(DE3) and existed mainly as inclusion bodies. The inclusion bodies were solubilized in urea and refolded by ion-exchange chromatography. After renaturation, the purity of the yielded rdhSCF was up to 90%. Cell proliferation assay showed that the specific activity of the rdhSCF was 2.86x10(5) U/mg, about 1.66 times as high as that of monomer rhSCF expressed in E. coli.     

基因重组大肠杆菌表达血红素加氧酶-1及培养条件优化

【背景】血红素加氧酶-1 (HO-1)具有抗氧化应激、抗凋亡和抗纤维化等多种生理效应,有望成为一种新型药物应用于临床疾病的治疗。【目的】构建表达HO-1的基因重组大肠杆菌(Escherichiacoli),并优化其表达培养条件,实现HO-1高产率的表达。【方法】PCR法克隆集胞藻(Synechocystissp.)PCC6803的HO-1基因(ho1),构建重组质粒pET-28a-ho1,转化大肠杆菌BL21(DE3)菌株,单因素实验优化表达培养基的种类、诱导剂添加时间、诱导培养时间、诱导剂浓度和诱导培养温度。【结果】构建了表达HO-1的基因重组大肠杆菌BL21(DE3)/pET-28a-ho1菌株,用甘油(GY)培养基培养至菌体浓度OD_(600)约为0.8时,加入终浓度为0.1 mmol/L的IPTG诱导,30°C诱导培养6 h,HO-1的表达量最高,Ni-NTA柱分离纯化得到的HO-1收率占细胞总蛋白的10.9%。【结论】获得了可溶性表达HO-1的基因重组大肠杆菌及其较佳的培养条件,为进一步研究集胞藻来源的HO-1的酶学性质和应用奠定了基础。     

Characteristics of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) production byRalstonia eutropha NCIMB 11599 and ATCC 17699

Ralstonia eutropha NCIMB 11599 and ATCC 17699 were grown, and their productions of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] compared. In flask cultures ofR. eutropha NCIMB 11599, cell concentration, P(3HB-co-4HB) concentration and polymer content decreased considerably with increases in the γ-butyrolactone concentration, and the 4HB fraction was also very low (maximum 1.74 mol%). In fed-batch cultures ofR. eutropha NCIMB 11599, glucose and γ-butyrolactone were fed as the carbon sources, under a phosphate limitation strategy. When glucose was fed as the sole carbon source, with its concentration controlled using an on-line glucose analyzer, 86% of the P(3HB) homopolymer was obtained from 201 g/L of cells. In a two-stage fed-batch culture, where the cell concentration was increased to 104 g/L, with glucose fed in the first step and constant feeding of γ-butyrolactone, at 6 g/h, in the second, final cell concentration at 67 h was 106 g/L, with a polymer content of 82%, while the 4HB fraction was only 0.7 mol%. When the same feeding strategy was applied to the fedbatch culture ofR. eutropha ATCC 17699, where the cell concentration was increased to 42 g/L, by feeding fructose in the first step and γ-butyrolactone (1.5 g/h) in the second, the final cell concentration, polymer content and 4HB fraction at 74 h were 51 g/L, 35% and 32 mol%, respectively. In summary,R. eutropha ATCC 17699 was better thanR. eutropha NCIMB 11599 in terms of P(3HB-co-4HB) production with various 4HB fractions.     

Expression and purification of the SsbB protein from Streptococcus pneumoniae

The Gram positive bacterium, Streptococcus pneumoniae, has two genes, designated ssbA and ssbB, which are predicted to encode single-stranded DNA binding proteins (SSB proteins). We have shown previously that the SsbA protein is similar in size and in biochemical properties to the well-characterized SSB protein from Escherichia coli. The SsbB protein, in contrast, is a smaller protein and has no counterpart in E. coli. This report describes the development of an expression system and purification procedure for the SsbB protein. The ssbB gene was amplified from genomic S. pneumoniae DNA and cloned into the E. coli expression vector, pET21a. Although, we had shown previously that the SsbA protein is strongly expressed from pET21a in the E. coli strain BL21(DE3)pLysS, no expression of the SsbB protein was detected in these cells. However, the SsbB protein was strongly expressed from pET21a in the Rosetta(DE3)pLysS strain, a derivative of BL21(DE3)pLysS which supplies the tRNAs for six codons that are used infrequently in E. coli. The differential expression of the two SSB proteins in the parent BL21(DE3)pLysS strain was apparently due to the presence of two rare codons in the ssbB gene sequence that are not present in the ssbA sequence. Using the Rosetta(DE3)pLysS/pETssbB expression system, a protocol was developed in which the SsbB protein was purified to apparent homogeneity. DNA binding assays confirmed that the purified SsbB protein had single-stranded DNA binding activity. The expression and purification procedures reported here will facilitate further investigations into the biological role of the SsbB protein.     

长双歧杆菌α-唾液酸苷酶在大肠杆菌中的表达及酶学性质

[背景]唾液酸苷酶是一类水解唾液酸糖复合物末端唾液酸残基的糖苷水解酶,广泛存在于动物和微生物中,具有重要的生物学功能.[目的]克隆一个长双歧杆菌(Bifidobacterium longum)唾液酸苷酶基因(blsia42)并在大肠杆菌(Escherichia coli)中表达,探讨该重组酶的酶学性质.[方法]从长双歧...     

High-level production of human leptin by fed-batch cultivation of recombinant Escherichia coli and its purification.

Human leptin is a 16-kDa (146-amino-acid) protein that is secreted from adipocytes and influences body weight homeostasis. In order to obtain high-level production of leptin, the human obese gene coding for leptin was expressed in Escherichia coli BL21(DE3) under the strong inducible T7 promoter. The recombinant leptin was produced as inclusion bodies in E. coli, and the recombinant leptin content was as high as 54% of the total protein content. For production of recombinant human leptin in large amounts, pH-stat fed-batch cultures were grown. Expression of leptin was induced at three different cell optical densities at 600 nm (OD600), 30, 90, and 140. When cells were induced at an OD600 of 90, the amount of leptin produced was 9.7 g/liter (37% of the total protein). After simple purification steps consisting of inclusion body isolation, denaturation and refolding, and anion-exchange chromatography, 144.9 mg of leptin that was more than 90% pure was obtained from a 50-ml culture, and the recovery yield was 41.1%.     

Optimization of an extracellular zinc-metalloprotease (SVP2) expression in Escherichia coli BL21 (DE3) using response surface methodology

In this work, SVP2 from Salinivibrio proteolyticus strain AF-2004, a zinc metalloprotease with suitable biotechnological applications, was cloned for expression at high levels in Escherichia coli with the intention of changing culture conditions to generate a stable extracellular enzyme extract. The complete ORF of SVP2 gene was heterologously expressed in E. coli BL21 (DE3) by using pQE-80L expression vector system. In initial step, the effect of seven factors include: incubation temperature, peptone and yeast extract concentration, cell density (OD600) before induction, inducer (IPTG) concentration, induction time, and Ca(2+) ion concentrations on extracellular recombinant SVP2 expression and stability were investigated. The primary results revealed that the IPTG concentration, Ca(2+) ion concentration and induction time are the most important effectors on protease secretion by recombinant E. coli BL21. Central composite design experiment in the following showed that the maximum protease activity (522 U/ml) was achieved in 0.0089 mM IPTG for 24h at 30 °C, an OD600 of 2, 0.5% of peptone and yeast extract, and a Ca(2+) ion concentration of 1.3 mM. The results exhibited that the minimum level of IPTG concentration along with high cell density and medium level of Ca(2+) with prolonged induction time provided the best culture condition for maximum extracellular production of heterologous protease SVP2 in E. coli expression system.