NDPK-A C4/109/145 S突变体降低磷酸转移酶活性但增加DNase活性

核苷二磷酸激酶A( nucleoside diphosphate kinase A, NDPK-A）有广泛的生物学活性,在肿瘤转移和调控中起重要作用.单独抑制NDPK-A中任何1个Cys所形成的二硫键,不会降低NDPK-A的磷酸转移酶活性和DNase活性.本实验通过构建C4/109/145S突变体并研究其生物学效应,为NDPK A结构与功能的研究提供参考.用定点突变法将NDPK-A 的4位、109位和145位Cys突变为Ser,构建pBV220-NDPK-A C4/109/145S和pEGFP-NDPK-A C4/109/145S两种重组质粒.在大肠杆菌中高效表达NDPK-A C4/109/145S突变体,纯化后可获得均一的重组NDPK-A C4/109/145S突变体蛋白.HPLC法和DNA消化法测定发现,C4/109/145S突变体磷酸转移酶活性低于野生型NDPK-A,而DNase活性高于野生型NDPK-A.以A549细胞作为模式细胞的流式细胞仪周期检测表明,C4/109/145S突变体与野生型NDPK-A一致,均可将细胞周期延滞在S期和G2/M期.这些结果证实,NDPK A结构异构与其磷酸转移酶活性密切相关,其酶活性至少需要1个Cys残基存在,NDPK-A结构中的二硫键也可能是其DNase活性的负调控机制之一,胞内NDPK-A的氧化还原异构可能对细胞周期无显著影响.     

人白细胞介素18半胱氨酸的定点突变及其对活性的影响

为研究IL 18结构与功能的关系 ,用重叠延伸PCR定点突变技术构建人白细胞介素 18(hIL 18) 4个半胱氨酸的突变体hIL 18C74 S、C10 4 S、C112 S和C163 S。将突变体的cDNA与原核细胞表达载体pJW2重组并转化大肠杆菌JM10 1。经热诱导后 ,4个突变体在大肠杆菌中均得到了高效表达。表达的蛋白质主要以包涵体的形式存在。包涵体经超声破碎 ,2mol/L尿素洗涤 ,8mol/L尿素溶解 ,SephadexG 10 0柱纯化后 ,纯度可达 90 %以上。以诱导人外周血单个核细胞 (PBMC)产生IFN γ的能力为指标检测复性突变体的活性。结果显示除C10 4 S外 ,其他 3个突变体的生物活性均低于野生型hIL 18,C74 S、C112 S和C163 S的活性分别是野生型hIL 18活性的 5 %、5 8%和11%。证明Cys74 、Cys163 为hIL 18诱导产生IFN γ的功能所必需     

NisinZ的定点突变及突变体性质的研究

以本实验室构建的含nisZ基因的质粒pHJ201为模板,采用定点突变技术将乳链菌肽Z分子中B环第8位Thr突变为Ser（T8S）、将第2位Dhb突变为Dha和第31位His突变为Lys（T2S/H31K）以及将第27位Asn突变为Lys和第31位His突变为Lys（N27K/H31K）,以pMG36e为载体,电击转化乳酸乳球菌（L.lactis）NZ9800进行表达。对表达产物性质的研究结果表明,3个突变体的抑菌谱和溶解度未发生变化,其抑菌活性略有下降,但它们的稳定性表现各不相同：N27K/H31K的稳定性与NisinZ几乎一致,而T8S和T2S/H31K的稳定性有明显提高,在pH9条件下100℃加热5min仍不丧失抑菌活性。     

重组链激酶及其三种突变体的活性研究

利用PCR技术从Streptococucspyogenes的基因组DNA中扩增了链激酶的编码基因ska,并进行了序列分析 ,利用基因删除及定点位变技术获得了删除了C-末端 42个氨基酸残基编码区的突变链激酶基因skaΔC42 ,第 5 9位Lys残基突变为Glu的突变链激酶基因skaK5 9E以及删除C-末端 42个氨基酸且第 5 9位Lys残基突变为Glu的突变链激酶基因skaΔC42K5 9E ,将ska及其三种突变体分别克隆到表达载体pET 1 5b上 ,构建分别表达野生型链激酶 (SK)、C-末端缺失 42个氨基酸残基的突变体 (SKΔC42 )、第 5 9位Lys残基突变为Glu的突变体 (SKK5 9E)及C-末端缺失 42个氨基酸且第 5 9位Lys残基突变为Glu突变体 (SKΔC42K5 9E)的表达载体pSK ,pSKΔC42 ,pSK K5 9E ,pSKΔC42K5 9E ,分别转化E .coliBL2 1 (DE3) ,IPTG诱导后在大肠杆菌中实现了高效表达 ,经亲和层析、离子交换层析及分子筛层析 ,获得了rSK、rSKΔC42、rSKK5 9E及rSKΔC42K5 9E ,活性分析表明rSK与其三种突变体具有相同的比活性。     

人白细胞介素18突变体的构建及其功能

为研究人白细胞介素 18(hIL 18)结构与功能的关系 ,用PCR定点突变技术分别构建了N末端、C末端缺失突变体(ΔNC)和IL 1特征样序列突变体S154A/Y156F/E157P/C163 T (S)。将突变体cDNA与原核表达载体 pJW 2重组并转化大肠杆菌DH5α ,经热诱导表达蛋白质 ,SDS PAGE证实表达的目的蛋白质以包涵体形式存在。菌体经超声破碎后 ,包涵体以 2mol/L尿素洗涤 ,8mol/L尿素溶解 ,并经SephadexG 75柱纯化 ,纯度可达 95 %以上。突变体蛋白质经逐步稀释复性后 ,以诱导人外周血单个核细胞 (PBMC)产生干扰素 γ(IFN γ)及对核因子 κB(NF κB)的激活能力为指标 ,检测突变体的生物学活性。结果显示ΔNC、S这 2个突变体对IFN γ的诱生能力显著低于野生型hIL 18,分别为野生型hIL 18的 13%和 4 8%。同时 ,ΔNC、S对NF κB的激活能力也低于野生型hIL 18,分别为野生型hIL 18的 6 9.7%和 89.8%。这些结果表明缺失的片段或突变的位点对hIL 18的功能有重要的作用。     

NisinZ的定点突变及突变体性质的研究

以本实验室构建的含nisZ基因的质粒pHJ2 0 1为模板 ,采用定点突变技术将乳链菌肽Z分子中B环第 8位Thr突变为Ser(T8S)、将第 2位Dhb突变为Dha和第 31位His突变为Lys(T2S H31K)以及将第 2 7位Asn突变为Lys和第 31位His突变为Lys(N2 7K H31K) ,以pMG36e为载体 ,电击转化乳酸乳球菌 (L .lactis)NZ980 0进行表达。对表达产物性质的研究结果表明 ,3个突变体的抑菌谱和溶解度未发生变化 ,其抑菌活性略有下降 ,但它们的稳定性表现各不相同 :N2 7K H31K的稳定性与NisinZ几乎一致 ,而T8S和T2S H31K的稳定性有明显提高 ,在pH9条件下10 0℃加热 5min仍不丧失抑菌活性。     

慢性乙型肝炎病毒耐药突变体研究

在治疗慢性乙型肝炎的核苷类似物的用药过程中,筛选耐药突变体.选定1名从未接受抗病毒治疗慢性乙型肝炎患者,用抗病毒核酸类药物治疗,不同治疗时期提取血清HBV DNA,用引物P1(5′-AAGGG-TATCTTGCCCGTTTGTCGTA-3′)和P2(5′-AAGCAGGATAGCCACAGA-3′)为第1轮,P3(5′-AAGGCACTTGTAT-TCCCATCCGAG-3′)和P4(5′-AAGGTCTATTTACAGGGGA-3′)为第2轮引物扩增筛选耐药突变体.在18周和22周分别检测到突变体LMV rtH69T(YMDDlocus)和LMV rtT184T(YMDDlocus)、LMV rtM204I(YMDDlocus).在60周和70周分别检测到ADV T213S、ADV T222A、ADV K212T和ADV S196L、ADV S242H,其中ADV S196L和ADV S242H 2种突变体是首次检测到.HBV核苷酸类似物耐药突变体筛选,对研究HBV耐药的分子机制有帮助.     

核苷二磷酸激酶A的定点突变及C4S突变体的制备和活性研究

目的:对核苷二磷酸激酶A(NDPK-A)二硫键异构的关键残基C4进行定点突变,构建、表达并纯化C4S突变体,测定其磷酸转移酶活性和DNase活性,研究二硫键异构对NDPK-A活性的影响。方法:以pBV220-nm23-H1质粒作为模板,通过设计合适的引物对NDPK-A进行定点突变,将第4位半胱氨酸突变为丝氨酸,构建NDPK-AC4S突变体;在大肠杆菌BL21中表达,DEAE-sepharose Fas tFlow与Cibacron Blue 3GA Sepharose CL-4B纯化目的蛋白,获得均一重组蛋白,纯度达到98%;DNA序列测定及重组蛋白的肽质量指纹图谱(PMF)分析均证明构建正确突变体;高效液相色谱法(HPLC)与DNA消化法分别测定野生型NDPK-A与C4S突变体的磷酸转移酶活性与DNase活性差异。结果:NDPK-AC4S突变体的磷酸基转移酶与DNase酶活性均高于野生型NDPK-A。结论:NDPK-A缺失二硫键后,活性增高。NDPK-A形成链内二硫键可能是其活性负调控模式之一。     

通过定点突变提高乳链菌肽对热及pH的稳定性

【目的】通过定点突变技术改变乳链菌肽(nisin)特定位置氨基酸,获得性质改善的nisin突变体,为扩大其应用范围提供依据。【方法】在抑菌谱扩大的nisin单突变体M21K nisinZ的基础上,对M21K nisZ基因第29位丝氨酸密码子进行定点突变;将其克隆至乳酸菌表达载体pMG36e,并在Lactococcus lactis NZ9800中进行表达;双突变体M21K/S29K nisinZ经分离纯化后检测其在抑菌活性、抑菌谱和稳定性等方面的变化。【结果】与单突变体M21K nisinZ及野生型nisinZ(wild-type,WT)相比,双突变体M21K/S29K nisinZ对指示菌的抑菌活性虽有所下降,但其对温度及pH值的稳定性有显著提高。同时其抑菌谱与M21K nisinZ相同,可抑制革兰氏阴性菌,扩大了WT的抑菌谱。【结论】通过改变nisin分子特定位置的氨基酸可以改善nisin分子的理化性质,有可能得到应用范围更广的nisin品种。     

Asp^126、Asp^130、Asp^134对人白介素18功能的影响

为研究人白细胞介素18(hIL-18)中一些氨基酸残基对IL－18功能的影响,用重叠延伸PCR定点诱变技术构建hIL-18突变体hIL-18D^126N、hIL-18D^130K、hIL-18D^134K。将突变体cDNA与原核表达载体pJW2重组并转化大肠杆菌DH5α。经热诱导表达,3个突变体占菌体总蛋白质的15％－31％以上。SDS－PAGE证实,表达的蛋白质以包含体形式存在。包含体经超声破坏,2mol/L尿素洗涤,8mol/L尿素溶解,Sephadex G-75柱纯化后,纯度均可达95％以上。Western印迹表明3个突变体与野生型hIL-8具有相同的免疫原性。纯化的突变体蛋白质经复性后,以诱导人外周血单个核细胞（PBMC）产生于干扰素（IFN－γ）的能力为指标检测其活性,结果显示3个突变体的生物学活性分别为野生型IL－18的32％、8％、10％,表明hIL-18中126、130、134位的天冬氨酸（Asp)对其功能是必需的。     

Interaction of ribosomal proteins S5, S6, S11, S12, S18 and S21 with 16 S rRNA

We have examined the effects of assembly of ribosomal proteins S5, S6, S11, S12, S18 and S21 on the reactivities of residues in 16 S rRNA towards chemical probes. The results show that S6, S18 and S11 interact with the 690-720 and 790 loop regions of 16 S rRNA in a highly co-operative manner, that is consistent with the previously defined assembly map relationships among these proteins. The results also indicate that these proteins, one of which (S18) has previously been implicated as a component of the ribosomal P-site, interact with residues near some of the recently defined P-site (class II tRNA protection) nucleotides in 16 S rRNA. In addition, assembly of protein S12 has been found to result in the protection of residues in both the 530 stem/loop and the 900 stem regions; the latter group is closely juxtaposed to a segment of 16 S rRNA recently shown to be protected from chemical probes by streptomycin. Interestingly, both S5 and S12 appear to protect, to differing degrees, a well-defined set of residues in the 900 stem/loop and 5'-terminal regions. These observations are discussed in terms of the effects of S5 and S12 on streptomycin binding, and in terms of the class III tRNA protection found in the 900 stem of 16 S rRNA. Altogether these results show that many of the small subunit proteins, which have previously been shown to be functionally important, appear to be associated with functionally implicated segments of 16 S rRNA.     

Positions of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit of Escherichia coli

Neutron scattering distance data are presented for 33 protein pairs in the 30 S ribosomal subunit from Escherichia coli, along with the methods used for measuring distances between its exchangeable components. When combined with prior data, these new results permit the positioning of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit, completing the mapping of its proteins by neutron scattering. Comparisons with other data suggest that the neutron map is a reliable guide to the quaternary structure of the 30 S subunit.     

Interaction of proteins S16, S17 and S20 with 16 S ribosomal RNA

We have used rapid chemical probing methods to examine the effect of assembly of ribosomal proteins S16, S17 and S20 on the reactivity of individual residues of 16 S rRNA. Protein S17 strongly protects a compact region of the RNA between positions 245 and 281, a site previously assigned to binding of S20. Protein S20 also protects many of these same positions, albeit more weakly than S17. Strong S20-dependent protections are seen elsewhere in the 5' domain, most notably at positions 108, and in the 160-200 and 330 loop regions. Enenpectedly, S20 also causes protection of several bases in the 1430-1450 region, in the 3' minor domain. In the presence of the primary binding proteins S4, S8 and S20, we observe a variety of effects that result from assembly of the secondary binding protein S16. Most strongly protected are nucleotides around positions 50, 120, 300 to 330 and 360 in the 5' domain, and positions 606 to 630 in the central domain. In addition, numerous nucleotides in the 5' and central domains exhibit enhanced reactivity in response to S16. Interestingly, the strength of the S20-dependent effects in the 1430-1450 region is attenuated in the presence of S4 + S8 + S20, and restored in the presence of S4 + S8 + S20 + S16. Finally, the previously observed rearrangement of the 300 region stem-loop that occurs during assembly is shown to be an S16-dependent event. We discuss these findings with respect to assignment of RNA binding sites for these proteins, and in regard to the co-operativity of ribosome assembly.     

Expression and purification of human ribosomal proteins S3, S5, S10, S19, and S26

The cDNAs for the human ribosomal proteins S3, S5, S10, S19, and S26 were introduced into a pET-15b vector and recombinant proteins containing an N-(His)(6)-fusion tag were expressed in high yields. To resolve the problem of frameshift during expression of S26 caused by the presence of tandem arginine codons in its mRNA that are rare in Escherichia coli, we substituted the rare AGA codon with the more frequent arginine codon (CGC) using a primer with this mutation for PCR amplification of S26 cDNA. All proteins were expressed mainly in the form of inclusion bodies and purified to homogeneity by metal affinity chromatography in one step (except for S3). Expression of the full-length S3 was accompanied by the formation of a low molecular weight polypeptide that was co-purified with S3 by metal affinity chromatography. Complete purification of S3 required an additional gel-filtration step. The proteins were refolded by stepwise dialysis. Both identity and purity of the proteins were confirmed by 2D PAGE. The proteins obtained could be used in a wide range of applications in biophysics, biochemistry, and molecular biology.     

Immunochemical accessibility of ribosomal protein S4 in the 30 S ribosome. The interaction of S4 with S5 and S12

The reactivity of protein S4-specific antibody preparations with 30 S ribosomal subunits and intermediates of in vitro subunit reconstitution has been characterized using a quantitative antibody binding assay. Anti-S4 antibody preparations did not react with native 30 S ribosomal subunits; however, they did react with various subunit assembly intermediates that lacked proteins S5 and S12. The inclusion of proteins S5 and S12 in reconstituted particles resulted in a large decrease in anti-S4 reactivity, and it was concluded that proteins S5 and S12 are primarily responsible for the masking of S4 antigenic determinants in the 30 S subunit. The effect of S5 and S12 on S4 accessibility is consistent with data from a variety of other approaches, suggesting that these proteins form a structural and functional domain in the small ribosomal subunit.     

Further evidence that the ribosomal 30S proteins S3, S5, S9, S11, S12, and S18 possess specific 16S RNA binding sites

Summary E. coli ribosomal 16S RNA preparted by an acetic acid-urea extraction technique individually binds, in addition to the seven established proteins, 6 new 30S ribosomal proteins (S3, S5, S9, S12, S18 and S11) (Hochkeppel et al., 1976). In this communication we demonstrate the site specificity of these proteins. Binding curves of the individual proteins with acetic acid-urea 16S RNA show that the binding of all six proteins to the RNA reaches a plateau at 0.3–0.97 copies per 16S RNA molecule. No significant binding of these proteins to classical phenol extracted 16S RNA is observed, with the exception of S13 which binds 0.2 copies of protein per molecule of 16S RNA. Specificity of binding of these proteins is also demonstrated in chase experiments. The site specificity of individual [³H]-labeled 30S proteins bound to 16S RNA is tested by the addition of non-radioactive 30S total protein to the reaction mixture.     

RNA-protein cross-linking in Escherichia coli 30S ribosomal subunits; determination of sites on 16S RNA that are cross-linked to proteins S3, S4, S5, S7, S8, S9, S11, S13, S19 and S21 by treatment with methyl p-azidophenyl acetimidate.

RNA-protein cross-links were introduced into E. coli 30S ribosomal subunits by treatment with methyl p-azidophenyl acetimidate. After partial nuclease digestion of the RNA moiety, a number of cross-linked RNA-protein complexes were isolated by a new three-step procedure. Protein and RNA analysis of the individual complexes gave the following results: Proteins S3, S4, S5 and S8 are cross-linked to the 5'-terminal tetranucleotide of 16S RNA. S5 is also cross-linked to the 16S RNA within an oligonucleotide encompassing positions 559-561. Proteins S11, S9, S19 and S7 are cross-linked to 16S RNA within oligonucleotides encompassing positions 702-705, 1130-1131, 1223-1231 and 1238-1240, respectively. Protein S13 is cross-linked to an oligonucleotide encompassing positions 1337-1338, and is also involved in an anomalous cross-link within positions 189-191. Protein S21 is cross-linked to the 3'-terminal dodecanucleotide of the 16S RNA.     

Immunoprecipitation of 70S, 50S and 30S ribosomes ofEscherichia coli

Antibodies were raised in rabbits against 70S ribosomes, 50S and 30S ribosomal subunits individually. Purified immunoglobulins from the antiserum against each of the above ribosomal entities were tested for their capabilities of precipitating 70S, 50S and 30S ribosomes. The observations revealed the following: (i) The antiserum (IgG) raised against 70S ribosomes precipitates 70S ribosomes completely, while partial precipitation is seen with the subunits, the extent of precipitation being more with the 50S subunits than with 30S subunits; addition of 50S subunits to the 30S subunits facilitates the precipitation of 30S subunits by the antibody against 70S ribosomes. (ii) Antiserum against 50S subunits has the ability to immunoprecipitate both 50S and 70S ribosomes to an equal extent. (iii) Antiserum against 30S subunits also has the property of precipitating both 30S and 70S ribosomes. The differences in the structural organisation of the two subunits may account for the differences in their immunoprecipitability.     

Interaction of ribosomal proteins, S6, S8, S15 and S18 with the central domain of 16 S ribosomal RNA

We have constructed complexes of ribosomal proteins S8, S15, S8 + S15 and S8 + S15 + S6 + S18 with 16 S ribosomal RNA, and probed the RNA moiety with a set of structure-specific chemical and enzymatic probes. Our results show the following effects of assembly of proteins on the reactivity of specific nucleotides in 16 S rRNA. (1) In agreement with earlier work, S8 protects nucleotides in and around the 588-606/632-651 stem from attack by chemical probes; this is supported by protection in and around these same regions from nucleases. In addition, we observe protection of positions 573-575, 583, 812, 858-861 and 865. Several S8-dependent enhancements of reactivity are found, indicating that assembly of this protein is accompanied by conformational changes in 16 S rRNA. These results imply that protein S8 influences a much larger region of the central domain than was previously suspected. (2) Protein S15 protects nucleotides in the 655-672/734-751 stem, in agreement with previous findings. We also find S15-dependent protection of nucleotides in the 724-730 region. Assembly of S15 causes several enhancements of reactivity, the most striking of which are found at G664, A665, G674, and A718. (3) The effects of proteins S6 and S18 are dependent on the simultaneous presence of both proteins, and on the presence of protein S15. S6 + S18-dependent protections are located in the 673-730 and 777-803 regions. We observed some variability in our results with these proteins, depending on the ratio of protein to RNA used, and in different trials using enzymatic probes, possibly due to the limited solubility of protein S18. Consistently reproducible was protection of nucleotides in the 664-676 and 715-729 regions. Among the latter are three of the nucleotides (G664, G674 and A718) that are strongly enhanced by assembly of protein S15. This result suggests that an S15-induced conformational change involving these nucleotides may play a role in the co-operative assembly of proteins S6 and S18.     

Positions of proteins S6, S11 and S15 in the 30 S ribosomal subunit of Escherichia coli

A map of the positions of 12 of the 21 proteins of the 30 S ribosomal subunit of Escherichia coli (S1, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12 and S15), based on neutron scattering, is presented and discussed. Estimates for the radii of gyration of these proteins in situ are also obtained. It appears that many ribosomal proteins have compact configurations in the particle.