内含肽在构建亲和纯化系统中的应用

内含肽是前体未成熟蛋白中的一段具有自我剪接功能的多肽链,在蛋白质纯化、蛋白质连接、环肽制备、蛋白标记以及生物传感器等方面广泛应用。本文综述了内含肽应用于蛋白质亲和纯化的发展历程,分别对层析型和非层析型内含肽纯化体系进行了分析和讨论,并总结了对控制内含肽断裂反应所进行的研究,为进一步改善内含肽介导蛋白质纯化提供依据和线索。     

鳖源胶原蛋白提取纯化及在生物材料中的应用

近年来,胶原蛋白因其良好的生物学性能在生物材料应用中得到越来越多的关注,为了建立一种快速高效的鳖源胶原蛋白纯化方法和探究其在生物材料中的应用价值,首先用Van Gieson染色法和苦味酸-天狼星红染色法观察裙边胶原纤维组织结构,发现裙边胶原纤维含量非常高且类型主要是Ⅰ型。采用不同截留分子量的透析袋对裙边胶原蛋白粗提液进行直接透析纯化,发现截留分子量为100 k Da的透析袋在透析48 h后对裙边胶原蛋白的纯化效果最好,SDS-PAGE检验显示几乎没有杂带。对裙边胶原蛋白生物学性能包括吸水性、体外降解性进行考察,其吸水力和持水力分别高达12.06 g/g和98.21%,且在72 h后被完全降解;对裙边胶原蛋白海绵的溶血性、皮肤致敏性、肝部创伤止血性、促创伤皮肤愈合性进行了研究,并和交联胶原进行比较,发现裙边胶原蛋白和交联胶原均不会造成SD大鼠溶血和皮肤过敏,二者均具有良好的止血效果,且裙边胶原蛋白能显著缩短创伤皮肤愈合时间,具有良好的促创伤皮肤愈合性,而交联胶原效果欠佳。本研究表明裙边胶原蛋白表现出了优良的生物学性能,在生物材料领域具有很大应用价值。     

Quantitation and pharmacokinetic modeling of therapeutic antibody quality attributes in human studies

A thorough understanding of drug metabolism and disposition can aid in the assessment of efficacy and safety. However, analytical methods used in pharmacokinetics (PK) studies of protein therapeutics are usually based on ELISA, and therefore can provide a limited perspective on the quality of the drug in concentration measurements. Individual post-translational modifications (PTMs) of protein therapeutics are rarely considered for PK analysis, partly because it is technically difficult to recover and quantify individual protein variants from biological fluids. Meanwhile, PTMs may be directly linked to variations in drug efficacy and safety, and therefore understanding of clearance and metabolism of biopharmaceutical protein variants during clinical studies is an important consideration. To address such challenges, we developed an affinity-purification procedure followed by peptide mapping with mass spectrometric detection, which can profile multiple quality attributes of therapeutic antibodies recovered from patient sera. The obtained data enable quantitative modeling, which allows for simulation of the PK of different individual PTMs or attribute levels in vivo and thus facilitate the assessment of quality attributes impact in vivo. Such information can contribute to the product quality attribute risk assessment during manufacturing process development and inform appropriate process control strategy.     

Stimulating effect of both 4’‐O‐methylnorbelladine feeding and temporary immersion conditions on galanthamine and lycorine production by Leucojum aestivum L. bulblets

Bulb cultures of Leucojum aestivum and L. aestivum ‘Gravety Giant’ were subcultured in medium containing the precursor 4’‐O‐methylnorbelladine (MN) at various concentrations [0 (control), 0.15 and 0.3 g/L]. The cultures were conducted in bioreactor RITA® and lasted for 15, 30, 40 and 50 days. The growth rate and the alkaloid accumulation in bulblets were studied. For this latter purpose, a purification method was developed. It comprised a highly selective solid phase extraction using on the one hand, UPTI‐CLEAN SI and SCX cartridges for plant extracts and on the other hand, 2H cartridges for culture media. Pure alkaloidal fractions were, thus, analyzed by LC‐ESI‐MS allowing the quantitative evaluation of galanthamine and lycorine from culture extracts. Precursor feeding along with temporary immersion conditions was found to significantly improve the accumulation of both galanthamine and lycorine. The maximal concentrations of galanthamine (0.81 mg/g DW) and lycorine (0.54 mg/g DW) in L. aestivum bulblets were reached, respectively, after 40 days of culture with 0.15 g/L of precursor and after 30 days of culture with 0.3 g/L of precursor. In L. aestivum ‘Gravety Giant’ bulb cultures, 0.3 g/L of precursor was the best condition for both galanthamine (0.6 mg/g DW after 50 days) and lycorine (1.13 mg/g DW after 30 days).     

反胶束法萃取核桃胰蛋白酶抑制剂及其抗虫研究

植物胰蛋白酶抑制因子对植物本身具有保护作用,能调节植物蛋白质的合成和分解,并具有抗虫作用。为明确核桃蛋白酶抑制剂对小菜蛾的抗虫效果,该研究采用AOT/异辛烷反胶束法和亲和层析法萃取及纯化核桃胰蛋白酶抑制剂,命名为WTI,并对其抑制活性和生物活性进行测试。迪克森作图法测得纯化的核桃胰蛋白酶抑制剂的抑制常数为2.1×10-9mol/L。抗虫试验表明,小菜蛾自1龄起至化蛹前的整个幼虫阶段中,核桃胰蛋白酶抑制剂均对其产生了较强的毒杀作用。尤其是在1-3龄期,效果最佳。与对照组相比,WTI能延迟小菜蛾的化蛹时间,降低其化蛹率。核桃胰蛋白酶抑制剂对小菜蛾有明显的毒杀作用,但不表现出明显的生长抑制作用,且一定浓度的核桃胰蛋白酶抑制剂能将小菜蛾扼杀在其化蛹之前,从而能够控制其繁殖。     

连续血液净化治疗烧伤脓毒症50 例临床疗效及安全性研究

目的:研究连续血液净化治疗烧伤脓毒症的临床疗效及安全性。方法:选取我院收治的烧伤脓毒症患者110例,随机分成观察组(n=50)和对照组(n=60)。对照组采取常规治疗,观察组采取持续性血液净化治疗。观察并比较两组患者治疗前后PaO_2,WBC,GLU,Na,BUN,Cr,TNF-α及IL-8水平变化、临床疗效及安全性。结果:与治疗前相比,观察组患者治疗后PaO_2,WBC,GLU,Na,BUN,Cr,TNF-α及IL-8水平均改善,且优于对照组,差异具有统计学意义(P0.05)。观察组呼吸机使用时间和ICU住院时间均短于对照组,差异具有统计学意义(P0.05)。观察组28 d内死亡率(12.00%)低于对照组(40.00%),差异具有统计学意义(P0.05)。结论:持续性血液净化治疗是烧伤脓毒症治疗的安全有效方式,可改善患者各项指标,缩短呼吸机使用以及住院时间,降低患者短时间内病死率,十分具有临床价值。     

大孔树脂分离纯化辣椒叶总黄酮的工艺研究

目的:筛选适合分离纯化辣椒叶总黄酮的一种大孔树脂,同时用响应面法进行优化得到最佳纯化工艺。方法:采用热回流法提取辣椒叶总黄酮,以吸附率和解吸率为考察指标,考察6种不同型号的大孔树脂(HPD100、HPD450、HPD600、HPD826、D101、AB-8)对辣椒叶总黄酮的吸附能力与解吸能力,确定最佳树脂。通过动态吸附解吸实验考察此树脂对辣椒叶总黄酮的最佳分离纯化工艺。结果:通过对辣椒叶总黄酮吸附分离性能的分析显示HPD600为最佳树脂,最优工艺为:上样浓度为10 mg/mL,上样量为10 mL,洗脱体积为4 BV,洗脱液流速为4 mL/min,洗脱液pH为7,依次用水、10%、30%乙醇冲洗树脂柱,50%乙醇为洗脱液。纯化后的黄酮纯度435.4 mg/g。结论:该方法简便,操作简单,对辣椒叶总黄酮的纯化效果较好。     

Expression and purification of the matrix protein of Nipah virus in baculovirus insect cell system

Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N‐terminally His‐tagged NiV M protein in insect cells. A time‐course study demonstrated that the highest yield of recombinant M protein (400–500 μg) was expressed from infected cells 3 days after infection. A single‐step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme‐linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:171–177, 2016     

Process integration for recovery of recombinant collagen type I α1 from corn seed

Because of safety concerns and product consistency issues with the use of animal‐derived collagen, several recombinant protein expression hosts have been considered for recombinant collagen corn seed. Full length, triple‐helical, recombinant collagen (rCIα1) is expressed as a fusion with a foldon domain, which must later be removed. Here we have examined integration of purification and foldon removal by comparing advantages of removal before or after purification, using salt precipitation as the main purification step. Because expression levels in available maize lines are low, Pichia‐produced recombinant collagens, both with and without foldon, were added to corn seed germ at the extraction step. Salt precipitation of an acidic corn seed extract yielded 100% of the collagen without foldon at >70% purity without the pepsin pretreatment. With pepsin pretreatment, yield was 94.0% with purity of 76.5%. Analysis of the protein molecular weight distribution of the pre‐ and post‐treatment extracts showed that the corn proteins are largely resistant to pepsin proteolysis, explaining why little benefit was obtained by pepsin treatment. In the absence of pepsin treatment, the recovery of rCIα1 with foldon was still above 90% but the purity was only 44%. This still represented at about 13‐fold purification with a 2.7‐fold volume reduction which would reduce the pepsin requirement for post‐recovery foldon cleavage. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:98–107, 2016