Manman Su

Expression, purification and characterization of a human single-chain Fv antibody fragment fused with the Fc of an IgG1 targeting a rabies antigen in Pichia pastoris.

Because the demand for rabies post exposure prophylaxis (PEP) treatment has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide an adequate amount of the required passive immune component in PEP in countries where canine rabies is endemic. The replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for the treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a human single-chain Fv antibody fragment fused with the Fc of an IgG1 targeting a rabies antigen to develop a product that can be used as a component of the PEP cocktail. We cloned the ScFv fragment from a human ScFv library that was established previously and inserted this fragment into the expression vector pPICZαC/Fc. An active recombinant ScFv-Fc fusion protein was successfully expressed in Pichia pastoris. The production of ScFv-Fc was optimized and scaled up in an 80L fermentor with yields exceeding 60mg/L. The ScFv-Fc protein was purified to more than 95% purity using a two-step scheme: ammonium sulfate fractionation and Protein A Sepharose CL-4B. The ScFv-Fc fusion protein neutralized rabies virus in a standard in vivo neutralization assay in which the virus was incubated with the ScFv-Fc molecules before intracranial inoculation in mice. Our results suggest that functional antibodies can be produced in P. pastoris and that ScFv-Fc fusion proteins have the potential to serve as therapeutic candidates.

Expression and purification of recombinant ATF-mellitin, a new type fusion protein targeting ovarian cancer cells, in P. pastoris.

Melittin is well known to possess cytolytic activity with wide-spectrum lytic properties and its potential use as an agent to treat several types of cancer has been tested. Due to the non-specific toxicity, melittin can impair not only cancer cells but also normal tissue. Thus, tumor-targeted toxins may be helpful for developing novel anticancer therapeutics. The urokinase-type plasminogen activator (uPA) plays a central role in tissue remodelling events occurring in normal physiology and in pathophysiology, including cancer invasion and metastasis. Heartening findings showed that uPA receptor is predominantly expressed on many types of cancer. Therefore, the amino-terminal fragment (ATF) of uPA which was able to identify and bond with cancer cells was used as the cell-targeting domain to make up tumor-targeted toxin in this study. In the present study, pPICZαC-ATF-melittin eukaryotic expression vector was successfully constructed. After transformed into P. pastoris and induced by methanol, rATF-mellitin was detected by SDS-PAGE and western blot analysis. After induction with methanol, the expression level of rATF-mellitin was 312 mg/l in 80-l fermentor. rATF‑mellitin was purified to >95% purity using SP Sepharose ion exchange chromatography and source™ 30 RPC with 67.2% recovery. Cell proliferation assay showed that rATF-melittin inhibited growth of SKOV3 cells and had no cytotoxicity effect on normal cells. For the first time, we established a stable and effective rATF-mellitin P. pastoris expression system to obtain a high level of expression of secreted rATF-mellitin which was purified by a highly efficient purification procedure.

Expression and anticancer activity analysis of recombinant human uPA1‑43-melittin.

The present study is focused on expression of a target fusion protein which can be used in ovarian cancer target therapy. It aimed to construct human urokinase-type plasminogen activator (uPA)(1-43)-melittin eukaryotic expression vector to express recombinant human uPA(1-43)-melittin (rhuPA(1-43)-melittin) in P. pastoris and to detect its anticancer effects on ovarian cancer cells. The DNA sequences that encode uPA1-43 amino acids and melittin were synthesized according to its native amino acid sequences and consequently inserted into pPICZαC vector. Then uPA1-43-melittin -pPICZαC was transformed into P. pastoris X-33, and rhuPA(1-43)-melittin was expressed by methonal inducing. The bioactivities of recombinant fusion protein were detected with inhibition effects on growth of ovarian cancer cells, cell cycle detection and TUNEL assay. The results of DNA sequence analysis of the recombinant vector uPA(1-43)-melittin -pPICZαC demonstrated that the DNA encoding human uPA 1-43 amino acids and 1-26 amino acids of melittin was correctly inserted into the pPICZαC vector. After being induced by methonal, fusion protein with molecular weight 7.6 kDa was observed on the basis of SDS-PAGE and western blot analysis. The recombinant protein was able to suppress growth of SKOV3, induce cell cycle arrest and apoptosis of SKOV3 cells. The fusion protein does not have any obvious toxicity on normal tissues. RhuPA(1-43)-melittin was successfully expressed in P. pastoris. Taking uPA(1-43) amino acids specifically binding to uPAR as targeted part of fusion protein, and making use of antitumor activity of melittin, the recombinant fusion protein it was able to inhibit growth of ovarian tumors and to be applied for effective targeted treatment.

Long non-coding RNAs on the stage of cervical cancer (Review)

Cervical cancer is one of most malignant gynecological tumors. However, effective means for diagnosing and treating cervical cancer have yet to be identified. A few decades ago, long non-coding RNAs (lncRNAs) were regarded as useless parts of the genome, however, increasing data have demonstrated the importance of lncRNAs in the diagnosis and treatment of cervical cancers. The aim of the present study is to summarize the role(s) of HOTAIR, MALAT1, CCAT2, SPRY4-IT1, RSU1P2, CCHE1, lncRNA-EBIC and PVT1. Approximately 14 lncRNAs are involved in cervical cancer and several important proteins, miRNAs and other molecules and play crucial roles in a few traditional signaling pathways that have been proven to be related to those lncRNAs. In conclusion, lncRNAs may be useful as exact treatment targets and diagnostic biomarkers for improving therapies in cervical cancer patients and lncRNAs may contribute to effective diagnosis and treatment methods for cervical cancer.

Expression and purification of recombinant human apolipoprotein C-I in Pichia pastoris

Apolipoprotein C-I (ApoC-I) is a small, basic apolipoprotein which is mainly secreted by the liver as a component of triglyceride-rich lipoproteins and high density lipoproteins whose importance in plasma lipoprotein metabolism is increasingly evident. At present, the only way to obtain native ApoC-I is separating it from human plasma. The methods have some restrictions on source, the complicated technology, the potential infections and a high cost which limits the research and application of native ApoC-I. Because of its small size, ApoC-I has previously been prepared by peptide synthesis which is also limited by a high cost. Therefore, in this study, a Pichia pastoris expression system was first used to obtain a high level expression of secreted, recombinant human ApoC-I (rhApoC-I).

High yield and purification of recombinant human apolipoprotein E3 in Pichia pastoris

Apolipoprotein E3 (ApoE3) is an important apolipoprotein in plasma and plays a critical role in lipid transport and cholesterol homeostasis. As the only natural source of this protein, human blood cannot provide large-scale ApoE3 for research and applications. Therefore, in our study, a Pichia pastoris expression system was first used to obtain a high-level expression of secreted, recombinant human ApoE3 (rhApoE3). The full-length sequence encoding ApoE3, gained by RT-PCR, was inserted into the pPICZalphaC vector and transformed into P. pastoris strain X33, and then the high expression transformants with zeocin resistance were obtained. The growth conditions of the transformant strains were optimized in 50ml conical tubes including pH and inducing time. After induction with methanol, the expression level of rhApoE3 was 120 mg/L in 80 L fermentor. RhApoE3 was purified more than 94% purity using SP Sepharose ion exchange chromatography and source 30RPC. A preliminary biochemical characterization of purified rhApoE3 was performed by analyzing the ability of inhibiting PDGF-induced proliferation of rat coronary artery smooth muscle cells (SMCs), and the results demonstrated that the function of purified rhApoE3 was similar to natural human ApoE3.

Expression and Purification of Recombinant Human Apolipoprotein A-II in Pichia pastoris

Apolipoprotein A-II (ApoA-II) is the second most abundant protein constituent of high-density lipoprotein (HDL). The physiologic role of ApoA-II is poorly defined. ApoA-II may inhibit lecithin:cholesterol acyltransferase and cholesteryl-ester-transfer protein activities, but may increase the hepatic lipase activity. ApoA-II may also inhibit the hepatic cholesteryl uptake from HDL probably through the scavenger receptor class B type I depending pathway. Interpretation of data from transgenic and knockout mice of genes involved in lipoprotein metabolism has been often complicated as clinical implications because of species difference. So it is important to obtain human ApoA-II for further studies about its functions. In our studies, Pichia pastoris expression system was first used to express a high-level secreted recombinant human ApoA-II (rhApoA-II). We have cloned the cDNA encoding human ApoA-II and achieved its high-level secreting expression with a yield of 65 mg/L of yeast culture and the purification process was effective and easy to handle. The purified rhApoA-II can be used to further study its biological activities.

Preparation and activity analysis of recombinant human high-density lipoprotein.

Population studies have consistently shown a highly inverse correlation between plasma concentration of high-density lipoprotein and the risk of atherosclerotic cardiovascular disease in humans. High-density lipoprotein (HDL) as a therapeutic target is an intense area of ongoing investigation. Aiming to solve the shortcomings of native HDL application, we prepared recombinant human HDL (rhHDL) that contains a similar composition and has similar functions with native HDL. Six kinds of recombinant human apolipoproteins (rhapo)-rhapoA-I, rhapoA-II, rhapoA-IV, rhapoC-I, rhapoC-II, and rhapoE-were expressed in Pichia pastoris and purified with chromatography. By the facilitation of cholate, six kinds of rhapo penetrated among the phosphatidylcholine acyl chains. After purification by density-gradient centrifugation, rhHDL was acquired. Based on morphological observation, we confirmed that the micellar complexes of rhapo with phosphatidylcholine and cholesterol were prepared. We carried on comparative studies in vitro and in vivo between native HDL and rhHDL. Cellular cholesterol efflux assays showed that rhHDL could promote the efflux of excess cholesterol from macrophages. Furthermore, rhHDL has similar effects with native HDL on the blood lipid metabolism in hyperlipidemic mice. In conclusion, rhHDL has similar effects on antiatherosclerosis with native HDL through reverse cholesterol transport, antioxidative, and antithrombotic properties. It could be used as a therapeutic HDL-replacement agent.