Heterologous protein expression in the methylotrophic yeast Pichia pastoris

During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of P. pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology; (2) the ability of P. pastoris to produce foreign proteins at high levels, either intracellularly or extracellularly; (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing; and (4) the availability of the expression system as a commercially available kit. In this paper, we review the P. pastoris expression system: how it was developed, how it works, and what proteins have been produced. We also describe new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.  

‘ATG vectors’ for regulated high-level expression of cloned genes in Escherichia coli

A plasmid cloning vector system has been constructed that allows for the production of large quantities of foreign proteins or fragments thereof, in an unfused state. These vectors provide strong regulated trp-lac fusion promoters and the lacZ ribosome-binding site (RBS) followed by an ATG translation initiation codon at an appropriate distance from the RBS. The ATG codon is located within a unique NcoI restriction site (CCATGG). Digestion with NcoI exposes the ATG for fusion. Gene fragments lacking a prokaryotic RBS and/or ATG start codons can be inserted in several ways. Expression experiments using a truncated cI gene of bacteriophage A or a large portion of the coding region of the Herpes simplex virus type l glycoprotein D gene have been performed. The results of these studies show that the vectors are useful for the high-level expression of prokaryotic and eukaryotic genes in Escherichia coli.  

A variation in the structure of the protein-coding region of the human p53 gene

An extensive analysis of genomic DNA preparations from a number of normal and malignant tissues revealed BglII site polymorphism of the human p53 gene. Approximately 10% of p53 gene alleles were found to contain an additional BglII site localized in a region of intron I. This allelic form of p53 gene was also responsible for p53 protein having altered electrophoretic mobility. Molecular cloning and sequencing of both the alleles of p53 gene revealed a base-pair change in codon 72 causing arginine → proline substitution in the allele with the additional BglII site. Both variants of the p53 gene may occur in homozygous state and are therefore functional.  

An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues

The type 2A protein phosphatases in mammalian tissue extracts are inhibited completely and specifically by 1–2 nM okadaic acid. In contrast, type 1 protein phosphatases are hardly affected at these concentrations, complete inhibition requiring 1 μM okadaic acid. These observations have been exploited to develop an improved procedure for the identification and quantitation of type 1, type 2A and type 2C protein phosphatases in tissue extracts.  

Endogenous mutagens and the causes of aging and cancer

A very large oxidative damage rate to DNA occurs as part of normal metabolism. In each rat cell the steady-state level is estimated to be about 10⁶ oxidative adducts and about 10⁵ new adducts are formed daily. It is argued that this endogenous DNA damage is a major contributor to aging and the degenerative diseases of aging, such as cancer. The oxidative damage rate in mammalian species with a high metabolic rate, short life span, and high age-specific cancer rate is much higher than the rate in humans, a long-lived creature with a lower metabolic rate and a lower age-specific cancer rate. It is argured that deficiency of micronutrients, such as dietary antioxidants or folate, is a major contributor to human cancer and degenerative diseases.

Understanding the role of mitogenesis in mutagenesis is critical for clarifying the mechanisms of carcinogenesis and interpreting high-dose animal cancer tests. High-dose animal cancer tests have been done mainly on synthetic industrial chemicals, yet almost all of the chemicals humans are exposed to are natural. About half of natural chemicals tested in high-dose animal cancer tests are rodent carcinogens, a finding that is consistent with the view that high-dose tests frequently increase mitogenesis rates. Animals have numerous defenses against toxins that make them very well buffered against low doses of almost all toxins, whether synthetic or natural.  

芦荟多糖的研究

本研究从芦荟(Aloe vera L.var.chinensis(Haw.)Berger)中分出三个多糖(A_(60) A_(90a)、和 A_(90b)分子量分别为12,000、47,000和12,000。除 A_(9Ca)的量过少,未作分析外,A_(60)的结构被推断为β-(1→4)连结的甘露聚糖,在2,3或6位上部分乙酰化;A_(90b)的结构被推断为β-(1→4)结构的直链葡萄-甘露聚糖。实验表明A_(60)对小鼠淋巴细胞转化功能和腹腔巨噬细胞的增殖均有一定的促进作用。因此,可作为抗肿癌的辅助药,在治疗免疫功能低下方面可能有应用价值。  

bcl—2基因的转录调控

许多不同的外界刺激，包括生长因子和细胞因子等都能诱导ｂｃｌ－２基因的表达，而且很显然，这种爱诱导表达的Ｂｃｌ－２对于细胞的存活是至关重要的。因此，为了对ｂｃｌ－２的转录调控研究有了初步的了解，将从转录水平和转录后水平两个层次上谈谈目前在该领域中的最新进展。