Cloning, expression and characterization of a HER2-alpha luffin fusion protein in Escherichia coli.

In recent decades, immunotoxins have attracted significant attention in treatment of a wide range of diseases including cancers due to their natural origins and their role in blocking crucial pathways within the cells. Ribosome inactivating proteins (RIPs) are efficient molecules in blocking protein synthesis through interactions with ribosomal rRNA molecules. cDNA molecule encoding HER2 scFv antibody fragment originated from trastuzumab attached to the mature alpha luffin gene fragment was subcloned into pET28a expression vector and expressed in different E. coli expression hosts. Identity of the expressed recombinant protein was investigated through western blotting and the fusion protein was purified using Ni-NTA affinity chromatography. The biological activity (toxicity) of the protein was investigated on DNA and RNA samples. A 58 kDa protein was expressed in E. coli. The best protein expression level was achieved in 0.2 mM IPTG at 30 °C in TB medium using E. coli BL21 (DE3) host strain. The fusion protein showed RNase and DNA glycosylase activity on tested RNA and DNA samples. DNA glycosylase activity of the recombinant fusion protein showed that alpha luffin part of this protein is active in conjugation to the scFv molecule and the expressed protein can be further studied in targeted biological in vitro assays.

Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein.

BACKGROUND: The increase of the protein expression via ribosomal manipulation is one of the suggested cellular mechanisms involved in EnBase fed-batch mode of cultivation. However, this system has not been implemented for cytotoxic proteins. OBJECTIVES: Here, the expression pattern of α-Luffin, a ribosome inactivation protein (RIP) with an innate toxicity, was investigated in EnBase system and the effect of low temperature cultivation on the increase of α-Luffin solubility was determined. MATERIALS AND METHODS: The encoding cDNA for mature α-Luffin was synthesized and subcloned into pET28a plasmid under the control of T7 promoter. The E. coli expression yield in EnBase® Flo fed-batch system was compared with traditional batch mode at two temperatures: 25 °C and 30 °C. Sampling was performed at several time intervals and solubility of recombinant-protein was checked on SDS-PAGE in pellet and supernatant samples. The purification of recombinant protein was performed by Ni-NTA column. RESULTS: In fed-batch cultivation mode, the early incubation time was desirable at 30 °C whereas the maximum amount of soluble α-Luffin was achieved from the extended protein synthesis period (12 and 24h post induction) at 25 °C. CONCLUSIONS: Our founding showed that EnBase had a greater efficacy in producing higher soluble protein ratios compared to batch cultivation growth rate, however for cytotoxic proteins, incubation temperature and time need to be optimized. Owing to the advantages of natural toxins from RIP family for producing anticancer immune-conjugates, well optimization of this protein expression is of importance regarding industrial aspects. The optimized condition proposed here is promising in terms of large scale soluble production of α-Luffin without the need for refolding.

In vivo measurement of Helicobacter pylori infection.

Helicobacter pylori is a well-recognized gastroduodenal pathogen (National Institute of Health Consensus Conference, JAMA 272:65-9, 1994) and a class I carcinogen (International Agency for Research on Cancer, IARC Monograph on the Evaluation of Carcinogenic Risk to Humans 61:177-240, 1994) which successfully colonizes the harsh acidic environment of the stomach. H. pylori is the causative factor for peptic ulcer disease (PUD) and an independent risk factor for gastric adenocarcinoma development. Therefore, accurate detection of infection is crucial for devising proper eradication regimens and preventing the more severe GI complications.Detection of H. pylori in the gastric mucosa can be performed via (1) direct detection of the bacterium; culture, histology and polymerase chain reaction (PCR) or (2) indirect detection of its enzymatic products particularly urease as well as serum H. pylori-specific antibody responses, which can be detected by rapid urease test (RUT) and serology, respectively.The accuracy of these diagnostic tests is reported as follows: 98.1% for bacterial culture, 98.1% for histology, 94.3% for PCR, 96.2% for RUT, and 84.9% for serology (Ni et al, J Pediatr 136(6):823-7, 2000).