专肽生物_定制多肽合成服务_多肽合成定制服务_多肽合成厂家_多肽合成公司

Definition

Bacterial peptides are protein fragments which are either part of a bacterium or produced by a bacteria1.

Classification

Different classes of peptides are produced by bacteria. Some examples include, antibiotics, enterotoxins, flagellar proteins, lipoproteins and various enzymes1.

Structural Characteristics

Structural characteristics of some bacterial peptides are described below-

A)      Malaria merozoite surface peptide (MSP-1): It is synthesized as a large precursor on the surface of the bacterium Plasmodium falciparum.  Proteolytic cleavage results in the production of a 19 KDa product whose tertiary structure is maintained by disulphide bridges2.

B)     Giardia variable surface protein: This peptide is the specific conserved region of the Giardia variable surface proteins (VSPs) that are cysteine rich zinc finger proteins. VSPs differ in size and sequence, they are characterized by this highly conserved C-terminal membrane spanning region, a hydrophilic cytoplasmic tail with a conserved five amino acid CRGKA signature sequence3,4.

C)    P.falciparum liver stage antigen 3: The protein is 200Kda and is highly conserved among parasites from different geographic regions5.

Mode of action

A)     MSP-1 is known to trigger antibody response by CD4 helper T cells. It is likely that these cells bind to the C-terminal domain of MSP-12.

B)     VSPs have a conserved hydrophilic amono acid trail that is palmitoyted by palmityl tranferases upon which they are activated3,4.

C)    P. falciparum liver stage antigen 3 is a potent antigen that is recongnized by T cells5.

Functions

A)     MSP-1 is a vaccine candidate for Plasmodium falciparum infection. It triggers a CD-4 T cell response2.

B)     VSPs are necessary for survival in the environment and host infection3,4.

C)    P.falciparum stage antigen 3 is also a good candidate vaccine as it activates both T and B cell responses5.

References

1.     Gitai Z (2005). "The new bacterial cell biology: moving parts and subcellular architecture". Cell, 120 (5): 577–86.

2.     Stuart JQ and Jean L (2001). Different regions of the malaria merozoite surface protein 1 of Plasmodium chabaudi elicit distinct T-cell and antibody isotype responses. Infect Immun, 69(4): 2245–2251.

3.     Davids BJ, Reiner DS, Birkeland SR, Preheim SP, Cipriano MJ, McArthur AG, Gillin FD (2006). A New Family of Giardial Cysteine-Rich Non-VSP Protein Genes and a Novel Cyst Protein. PLoS ONE, 20,1:e44.

4.     Touz MC, Conrad JT, Nash TE (2005). A novel palmitoyl acyl transferase controls surface protein palmitoylation and cytotoxicity in Giardia lamblia. Mol Microbiol., 58 (4), 999-1011.

5.     Jean-Pierre S, Blanca LP, Karima B, Pierra D, Pierra D (2001). DNA Immunization by Plasmodium falciparum liver-stage antigen 3 induces protection against Plasmodium yoelii Sporozoite challenge. Infect Immun., 69, 1202–1206.

Ref: Tomb, JF. et al. Nature 388, 539 (1997).

多肽H2N-Ser-Lys-Ile-Gly-Lys-Val-NH2的合成步骤：

1、合成MBHA树脂：取若干克的MBHA树脂（如初始取代度为0.5mmol/g）和1倍树脂摩尔量的Fmoc-Linker-OH加入到反应器中，加入DMF，搅拌使氨基酸完全溶解。再加入树脂2倍量的DIEPA，搅拌混合均匀。再加入树脂0.95倍量的HBTU，搅拌混合均匀。反应3-4小时后，用DMF洗涤3次。用2倍树脂体积的10%乙酸酐/DMF 进行封端30分钟。然后再用DMF洗涤3次，甲醇洗涤2次，DCM洗涤2次，再用甲醇洗涤2次。真空干燥12小时以上，得到干燥的树脂{Fmoc-Linker-MHBA Resin}，测定取代度。这里测得取代度为 0.3mmol/g。结构如下图：

2、脱Fmoc：取1.7g的上述树脂，用DCM或DMF溶胀20分钟。用DMF洗涤2遍。加3倍树脂体积的20%Pip/DMF溶液，鼓氮气30分钟，然后2倍树脂体积的DMF 洗涤5次。得到 H2N-Linker-MBHA Resin 。（此步骤脱除Fmoc基团，茚三酮检测为蓝色，Pip为哌啶）。结构图如下：

3、缩合：取1.53mmol Fmoc-Val-OH 氨基酸，加入到上述树脂里，加适当DMF溶解氨基酸，再依次加入3.06mmol DIPEA，1.45mmol HBTU。反应30分钟后，取小样洗涤，茚三酮检测为无色。用2倍树脂体积的DMF 洗涤3次树脂。（洗涤树脂，去掉残留溶剂，为下一步反应做准备）。得到Fmoc-Val-Linker-MBHA Resin。氨基酸：DIPEA：HBTU：树脂=3：6：2.85：1（摩尔比）。结构图如下：

4、依次循环步骤二、步骤三，依次得到

H2N-Val-Linker-MBHA Resin

Fmoc-Lys(Boc)-Val-Linker-MBHA Resin

H2N-Lys(Boc)-Val-Linker-MBHA Resin

Fmoc-Gly-Lys(Boc)-Val-Linker-MBHA Resin

H2N-Gly-Lys(Boc)-Val-Linker-MBHA Resin

Fmoc-Ile-Gly-Lys(Boc)-Val-Linker-MBHA Resin

H2N-Ile-Gly-Lys(Boc)-Val-Linker-MBHA Resin

Fmoc-Lys(Boc)-Ile-Gly-Lys(Boc)-Val-Linker-MBHA Resin

H2N-Lys(Boc)-Ile-Gly-Lys(Boc)-Val-Linker-MBHA Resin

Fmoc-Ser(tBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Val-Linker-MBHA Resin

以上中间结构，均可在专肽生物多肽计算器-多肽结构计算器中，一键画出。

最后再经过步骤二得到 H2N-Ser(tBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Val-Linker-MBHA Resin，结构如下：

5、切割：6倍树脂体积的切割液（或每1g树脂加8ml左右的切割液），摇床摇晃 2小时，过滤掉树脂，用冰无水乙醚沉淀滤液，并用冰无水乙醚洗涤沉淀物3次，最后将沉淀物放真空干燥釜中，常温干燥24小试，得到粗品H2N-Ser-Lys-Ile-Gly-Lys-Val-NH2。结构图见产品结构图。

切割液选择：1）TFA：H2O=95%：5%

2）TFA：H2O：TIS=95%：2.5%：2.5%

3）三氟乙酸：茴香硫醚：1，2-乙二硫醇：苯酚：水=87.5%：5%：2.5%：2.5%：2.5%

（前两种适合没有容易氧化的氨基酸，例如Trp、Cys、Met。第三种适合几乎所有的序列。）

6、纯化冻干：使用液相色谱纯化，收集目标峰液体，进行冻干，获得蓬松的粉末状固体多肽。不过这时要取小样复测下纯度 是否目标纯度。

7、最后总结：

杭州专肽生物技术有限公司（ALLPEPTIDE https://www.allpeptide.com）主营定制多肽合成业务，提供各类长肽，短肽，环肽，提供各类修饰肽，如：荧光标记修饰（CY3、CY5、CY5.5、CY7、FAM、FITC、Rhodamine B、TAMRA等），功能基团修饰肽（叠氮、炔基、DBCO、DOTA、NOTA等），同位素标记肽（N15、C13），订书肽（Stapled Peptide）,脂肪酸修饰肽（Pal、Myr、Ste），磷酸化修饰肽（P-Ser、P-Thr、P-Tyr），环肽（酰胺键环肽、一对或者多对二硫键环），生物素标记肽，PEG修饰肽，甲基化修饰肽

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